Quantification of biomarkers present in physiological samples

ABSTRACT

The present disclosure relates to immunoassays for NF-L, GFAP, UCH L1, and Tau performed on liquid samples derived from physiological fluids such as venous blood to detect the presence or absence of a physiological condition by quantifying one or a combination of NF-L, GFAP, UCH L1, and Tau at concentrations indicative of the condition.

CROSS REFERENCE TO RELATED APPLICATION

This application is a U.S. National Stage Application under 35 U.S.C. §371 of International Patent Application No. PCT/US2019/026640, filedApr. 9, 2019, which claims the benefit of U.S. Provisional ApplicationNo. 62/789,067, filed Jan. 7, 2019, and U.S. Provisional Application No.62/655,738, filed Apr. 10, 2018, the disclosure of each of which isincorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The present disclosure relates to multiplex immunoassays for quantifyinga plurality of biomarkers present in a physiological sample to detectthe presence or absence of a physiological condition.

BACKGROUND OF THE INVENTION

Recent advances in digital and spotted well immunoassay technologies areclosing in on next-generation capabilities to rapidly diagnosis seriousphysiological conditions that, even today, frequently go undiagnosed anduntreated with potentially tragic consequences. As but one example,digital immunoassays have been shown, at least in principle, to quantifysubtle changes in biomarkers indicative of traumatic brain injury (TBI)at very low concentrations that elude most other assay technologies. Abrain injury in a human may be caused by any number of events orconditions. In some cases, a brain injury may be caused by externalmechanical force, such as rapid acceleration or deceleration, impact,blast waves, or penetration by a projectile. This type of acquired braininjury is generally known as TBI. In the United States, more than 2.5million people seek medical care for TBI each year. Nonetheless, as of2015, no therapeutic has been approved by the U.S. Food and DrugAdministration to treat acute TBI, due, at least in part, to theinability to precisely diagnose TBI.

Digital and spotted well immunoassays have also shown potential fordetection of antigens indicative of crippling neurodegenerativedisorders. For example, neurofilament light chain has the potential fordetection of multiple sclerosis which affects more than 350,000 peoplein the U.S. and 2.5 million worldwide. In the U.S., prevalence estimatesvary between 5 and 119 per 100,000 and healthcare costs are estimated tobe more than $10 billion annually. It is the most common neurologicaldisease in young adults, with the risk of subsequent chronic functionalimpairment and disability after 10-15% of disease duration. While aphysician may diagnose multiple sclerosis in some patients soon afterthe onset of the illness, in other cases doctors may not be able toreadily identify the cause of the symptoms, leading to years ofuncertainty and multiple diagnoses. Unfortunately, no single laboratorytest is yet available to prove or rule out multiple sclerosis.

Certain existing methods and kits directed to measuring biomarkersrelevant to brain injury fail to target the correct biomarkers or failto include a sufficient number of biomarkers for providing relevantinformation about the presence or severity of these conditions. Also,many conventional assays lack the sensitivity to determine levels ofpotential clinical relevance. Accordingly, improved methods, tests,assays, kits, and systems for measuring biomarkers relevant to suchconditions are needed.

BRIEF SUMMARY OF THE INVENTION

Certain embodiments may provide, for example, a test for a neurologicalcondition (for example a neural injury, defect, disorder, or disease) ina subject. In certain embodiments, for example, the test may compriseproviding a liquid sample derived from a sample of physiological fluid.In certain embodiments, for example, the test may comprise obtaining,via a single multiplex immunoassay (for example a digital multipleximmunoassay or a multiplex spotted well immunoassay), concentrations ofneurofilament light chain (NF-L), glial fibrillary acidic protein(GFAP), ubiquitin carboxyl-terminal hydrolase L1 (UCH L1), and Tau inthe liquid sample. In certain embodiments, for example, the test maycomprise calculating at least one classification value based on amultivariate classification model using the concentrations as inputs tothe multivariate classification model. In certain embodiments, forexample, the test may comprise assigning a risk (for example a risk ofoccurrence or presence) of the neurological condition, comprising:comparing the at least one classification value to at least onethreshold value.

A. In certain embodiments, for example, the multivariate classificationmodel may be a function of at least two biomarker concentrations, the atleast two biomarkers selected from the group consisting of NF-L, GFAP,UCH L1, and Tau. In certain embodiments, for example, the multivariateclassification model may be further a function of at least onedemographic parameter (for example age, gender, and/or ethnicity). Incertain embodiments, for example, the multivariate classification modelmay be a logistic regression. In certain embodiments, for example, themultivariate classification model may be a neural network. In certainembodiments, for example, the multivariate classification model mayprovide a receiver operating characteristic (ROC) curve having an areaunder the curve (AUC) of at least 0.7 (for example an AUC of at least0.8 or at least 0.9). In certain embodiments, for example, themultivariate classification model may be characterized by at least onep-value (for example at least one p-value of less than 0.01, less than0.001, or less than 0.0001).

In certain embodiments, for example, the multivariate classificationmodel may require a baseline concentration of at least one of NF-L,GFAP, UCH L1, and Tau from the subject (for example a baselineconcentration obtained from the subject prior to occurrence or suspectedoccurrence of the neurological condition) as an input to themultivariate classification model. In certain embodiments, for example,the classification value may be based at least on the concentrations ofat least three biomarkers selected from the group consisting of NF-L,GFAP, UCH L1, and Tau. In certain embodiments, for example, one value ofthe at least one classification value may comprise a ratio of theconcentrations of a first biomarker and a second biomarker determinedfrom the single multiplex immunoassay, the first biomarker and thesecond biomarker selected from the group consisting of NF-L, GFAP, UCHL1, and Tau. In certain embodiments, for example, the at least oneclassification value may comprise a ratio of the concentration of afirst biomarker selected from the group consisting of NF-L, GFAP, UCHL1, and Tau and a second biomarker selected from the group consisting ofNF-L, GFAP, UCH L1, and Tau.

In certain embodiments, for example, the test may further comprise:normalizing one or more of the concentrations based at least on a sampleage of the physiological fluid. In certain embodiments, for example, thetest may further comprise: normalizing one or more of the concentrationsbased at least on a sample size of the sample of physiological fluid. Incertain embodiments, for example, the test may further comprise:normalizing one or more of the concentrations based at least on sampleage of the sample of physiological fluid. In certain embodiments, forexample, the test may further comprise: normalizing one or more of theconcentrations based at least on one or more demographic characteristicsof a subject from which the sample of physiological fluid was taken. Incertain embodiments, for example, the one or more demographiccharacteristics may comprise age. In certain embodiments, for example,the one or more demographic characteristics may comprise ethnicity. Incertain embodiments, for example, the one or more demographiccharacteristics may comprise gender.

B. In certain embodiments, for example, a low risk of the neurologicalcondition may be assigned if the at least one classification value isless than at least one threshold value (for example if the at least oneclassification value is a plurality of classification values (forexample 2 classification values, 3 classification values, 4classification values, or more than 4 classification values) that havelower values than corresponding threshold values for each of theplurality of classification values). In certain embodiments, forexample, the method may further comprise: assigning an indeterminaterisk of the neurological condition if one of the at least oneclassification value exceeds the at least one threshold value.

C. In certain embodiments, for example, the test may further comprise:indicating a neuroimaging study if the at least one classification valueis greater than the at least one threshold value. In certainembodiments, for example, the test may further comprise: indicating aneuroimaging study if the at least one classification value is less thanthe at least one threshold value. In certain embodiments, for example,the test may further comprise: indicating subject observation if the atleast one classification value is greater than the at least onethreshold value. In certain embodiments, for example, the test mayfurther comprise: indicating subject observation without a neuroimagingstudy if the at least one classification value is greater than a firstvalue of the at least one threshold value and the at least oneclassification value is less than a second value of the at least onethreshold value. In certain embodiments, for example, the test mayfurther comprise: indicating a change in a course of therapy (forexample as an indication of a change in the neurological condition) ifthe at least one classification value is greater than the at least onethreshold value. In certain embodiments, for example, the test mayfurther comprise: indicating a change of therapy treatment (for exampleas an indication of progress of the neurological condition) if the atleast one classification value is less than the at least one thresholdvalue.

D. In certain embodiments, for example, the sample of physiologicalfluid may be obtained from a subject within 24 hours after a medicalprocedure is performed on a subject. In certain embodiments, forexample, the physiological fluid may be venous blood. In certainembodiments, for example, the sample of physiological fluid may not beused to derive the liquid sample until after an initial diagnosis of theneurological condition. In certain embodiments, for example, the sampleof physiological fluid may be taken from a subject while the subject isunder continual care of one or more healthcare providers during andfollowing a medical procedure.

E. In certain embodiments, for example, the neurological condition maybe a TBI. In certain embodiments, for example, the neurologicalcondition may be an acquired brain injury. In certain embodiments, forexample, the neurological condition may be collateral to trauma. Incertain embodiments, for example, the neurological condition may becollateral to ischemia. In certain embodiments, for example, theneurological condition may be collateral to toxic exposure. In certainembodiments, for example, the neurological condition may be collateralto neurological disease. In certain embodiments, for example, theneurological condition may be collateral to heart attack. In certainembodiments, for example, the neurological condition may be collateralto child birth. In certain embodiments, for example, the neurologicalcondition may be collateral to oxygen deprivation. In certainembodiments, for example, the neurological condition may be collateralto a vehicular accident. In certain embodiments, for example, theneurological condition may be collateral to a fall. In certainembodiments, for example, the neurological condition may be collateralto an assault. In certain embodiments, for example, the neurologicalcondition may be collateral to being struck by an object. In certainembodiments, for example, the neurological condition may beneurodegenerative disease. In certain embodiments, for example, theneurodegenerative disease may be a multiple sclerosis (MS) (for examplerelapse-remitting MS, primary progressive MS, progressive relapsing MS,and/or secondary progressive MS). In certain embodiments, for example,the neurodegenerative disease may be an Alzheimer's disease.

F. In certain embodiments, for example, the test may be indicated byindependent evidence of the neurological condition. In certainembodiments, for example, the test may be indicated by a lawsuitalleging the neurological condition. In certain embodiments, forexample, the test may be performed in conjunction with (or indicated by)a positive computerized tomography (CT) scan. In certain embodiments,for example, the test may be performed in conjunction with (or indicatedby) a positive MRI scan. In certain embodiments, for example, the testmay be performed after an initial diagnosis of the neurologicalcondition.

G. In certain embodiments, for example, the single multiplex immunoassaymay be a digital assay. In certain embodiments, for example, the singlemultiplex immunoassay may be a multiplex spotted well assay.

Certain embodiments may provide, for example, a single-sample test for aneurological condition (for example a TBI or MS). In certainembodiments, for example, the single-sample test may comprise providinga liquid sample derived from a single sample of physiological fluid froma subject. In certain embodiments, for example, the single-sample testmay comprise obtaining, via at least one immunoassay, concentrations ofNF-L, GFAP, UCH L1, and Tau in the liquid sample. In certainembodiments, for example, the single-sample test may comprise assigninga risk of the neurological condition in the subject. In certainembodiments, for example, the assigning a risk of the neurologicalcondition in the subject may comprise determining at least one measureof significance of differences between the concentrations of NF-L, GFAP,UCH L1, and Tau in the liquid sample and concentrations of NF-L, GFAP,UCH L1, and Tau in a group of healthy donors.

A. In certain embodiments, for example, the subject may be a neonate. Incertain embodiments, for example, the subject may be a child. In certainembodiments, for example, the subject may be a toddler. In certainembodiments, for example, the subject may be a teenager. In certainembodiments, for example, the subject may be an adult. In certainembodiments, for example, the subject may be at least 50 years old (forexample at least 60 years old, at least 70 years old, or at least 80years old).

B. In certain embodiments, for example, the single sample ofphysiological fluid may be obtained within 12 hours of an eventsuspected of causing the neurological condition in the subject. Incertain embodiments, for example, the single sample of physiologicalfluid may be obtained within 24 hours of an event suspected of causingthe neurological condition in the subject.

C. In certain embodiments, for example, the at least one measure ofsignificance of differences may be derived from a classification model(for example a statistical model). In certain embodiments, for example,the classification model may be a function of (or utilize as an input)concentrations for at least one biomarker in the liquid sample and inthe group of healthy donors, the at least one biomarker selected fromthe group consisting of NF-L, GFAP, UCH L1, and Tau. In certainembodiments, for example, the classification model may be further afunction of (or utilize as an input) at least one demographic parameter(for example age, gender, and/or ethnicity). In certain embodiments, forexample, the classification model may be a logistic regression. Incertain embodiments, for example, the classification model may be aneural network. In certain embodiments, for example, the classificationmodel may provide an ROC curve having an AUC of at least 0.7. In certainembodiments, for example, the determining may comprise calculating atleast one classification value based on a classification model; andcomparing the at least one classification value to at least onethreshold value. In certain embodiments, for example, the determiningmay comprise computing a statistical measure, the statistical measuremay comprise a statistic obtained from an analysis of variance (ANOVA).In certain embodiments, for example, the ANOVA may be a one-way ANOVA.In certain embodiments, for example, the ANOVA may be a one-way ANOVA onranks. In certain embodiments, for example, the ANOVA may benon-parametric. In certain embodiments, for example, the ANOVA maycomprise a Kruskal-Wallis test. In certain embodiments, for example, theANOVA may comprise a Mann-Whitney test. In certain embodiments, forexample, the at least one measure of significance of differences may bebased on at least three differences between the concentrations of NF-L,GFAP, UCH L1, and Tau in the liquid sample and concentrations of NF-L,GFAP, UCH L1, and Tau in a group of healthy donors.

Certain embodiments may provide, for example, a protocol indicated bypotential neurological condition (for example a potential neural injury,potential defect, potential disorder, or potential disease). In certainembodiments, for example, the protocol indicated by the potentialneurological condition may comprise a CT scan positive for theneurological condition. In certain embodiments, for example, theprotocol may further comprise by one of the single-sample tests for theneurological condition disclosed herein if the CT scan is positive forthe neurological condition. In certain embodiments, for example, theprotocol may be exclusive of a magnetic resonance imaging (MRI) scan. Incertain embodiments, for example, the protocol may be a modification ofanother protocol to replace an MRI scan with one of the single-sampletests for the neurological condition disclosed herein.

Certain embodiments may provide, for example, a modified protocolindicated by a potential neurological condition. In certain embodiments,for example, the modified protocol indicated by the potentialneurological condition may comprise a protocol for detection of aneurological condition comprising a CT scan and in which an MRI scan isreplaced by one of the single-sample tests for the neurologicalcondition disclosed herein. In certain embodiments, for example, the atleast one immunoassay may be a digital assay. In certain embodiments,for example, the at least one immunoassay may be a multiplex spottedwell assay. In certain embodiments, for example, the physiological fluidmay be derived from venous blood. In certain embodiments, for example,the physiological fluid may be a serum. In certain embodiments, forexample, the physiological fluid may be a plasma. In certainembodiments, for example, the physiological fluid may be whole blood.

Certain embodiments may provide, for example, a single-assay test for aneurological condition (for example a neural injury, defect, disorder,or disease). In certain embodiments, for example, the single-assay testmay comprise providing at least one liquid sample derived from a singlesample of physiological fluid from a subject. In certain embodiments,for example, the single-assay test may comprise obtaining, via animmunoassay, concentrations of NF-L, GFAP, UCH L1, and Tau in the atleast one liquid sample. In certain embodiments, for example, thesingle-assay test may comprise assigning a risk of the neurologicalcondition (for example a TBI or MS) in the subject, comprising:determining at least one measure of significance of differences betweenthe concentrations of NF-L, GFAP, UCH L1, and Tau in the liquid sampleand concentrations of NF-L, GFAP, UCH L1, and Tau in a group of healthydonors. In certain embodiments, for example, the immunoassay may be amultiplex digital immunoassay. In certain embodiments, for example, theimmunoassay may be a multiplex spotted well assay.

Certain embodiments may provide, for example, a dual-sample test for aneurological condition. In certain embodiments, for example, thedual-sample test may comprise providing, from a subject: a) a firstliquid sample derived from a sample of a first physiological fluid takenat a first time; and b) a second liquid sample derived from a sample ofa second physiological fluid taken at a second time. In certainembodiments, for example, the dual-sample test may comprise obtaining,via immunoassay, concentrations of NF-L, GFAP, UCH L1, and Tau in thefirst liquid sample and in the second liquid sample. In certainembodiments, for example, the dual-sample test may comprise assigning arisk of the neurological condition in the subject, comprising:determining at least one measure of significance of differences betweenthe concentrations of NF-L, GFAP, UCH L1, and Tau in the first liquidsample and the concentrations of NF-L, GFAP, UCH L1, and Tau in thesecond liquid sample.

A. In certain embodiments, for example, the second time may be laterthan the first time. In certain embodiments, for example, the first timemay be within 3 hours (for example within 6 hours, within 12 hours,within 1 day, or within 7 days) of an event suspected of causing anoccurrence of the neurological condition. In certain embodiments, forexample, the second time may be at least 2 hours (for example at least12 hours, at least 1 day, at least 7 days, at least 8 days, at least 9days, at least 10 days, at least 11 days, or at least 12 days) after thefirst time. In certain embodiments, for example, the second time may beat least 2 hours (for example at least 12 hours, at least 1 day, atleast 7 days, at least 8 days, at least 9 days, at least 10 days, atleast 11 days, or at least 12 days) after an event suspected of causingan occurrence of the neurological condition.

Certain embodiments may provide, for example, a dual-sample test for aneurological condition (for example a neural injury, defect, disorder,or disease). In certain embodiments, for example, the dual-sample testmay comprise providing, from a subject: a) a first liquid sample derivedfrom a first physiological fluid sample taken at a first time; and b) atleast a second liquid sample derived from at least a secondphysiological fluid sample taken at least a second time. In certainembodiments, for example, the dual-sample test may comprise obtaining,via immunoassay, concentrations of NF-L, GFAP, UCH L1, and Tau in thefirst liquid sample and in the at least the second liquid sample. Incertain embodiments, for example, the dual-sample test may compriseassigning a risk of the neurological condition (for example a TBI or aMS) in the subject, comprising: determining at least one measure ofsignificance of differences between the concentrations of NF-L, GFAP,UCH L1, and Tau in the first liquid sample and the concentrations NF-L,GFAP, UCH L1, and Tau in the at least the second liquid sample.

Certain embodiments may provide, for example, a method to distinguishbetween types of neurological conditions (for example a neural injury,defect, disorder, or disease). In certain embodiments, for example, themethod may comprise providing a liquid sample derived from a sample ofphysiological fluid from a subject. In certain embodiments, for example,the method may comprise obtaining, via at least one immunoassay,concentrations of NF-L, GFAP, UCH L1, and Tau in the liquid sample. Incertain embodiments, for example, the method may comprise distinguishingbetween types of neurological conditions, comprising: a) classifying asstatistically significant a difference between at least a firstbiomarker concentration of the concentrations of NF-L, GFAP, UCH L1, andTau in the liquid sample and concentrations of NF-L, GFAP, UCH L1, andTau in a group of healthy donors; and b) determining that a differencebetween at least a second biomarker concentration of the concentrationsof the NF-L, GFAP, UCH L1, and Tau in the liquid sample and theconcentrations of NF-L, GFAP, UCH L1, and Tau in the group of healthydonors is statistically insignificant.

A. In certain embodiments, for example, the method may distinguishbetween mild TBI and severe TBI. In certain embodiments, for example,the at least a first biomarker may comprise GFAP and the at least afirst biomarker may be exclusive of NF-L. In certain embodiments, forexample, the at least a second biomarker may comprise NF-L.

In certain embodiments, for example, the method may distinguish betweena neurological condition arising from isolated contusion (or diffuseaxonal injury) and at least one other type of neurological condition. Incertain embodiments, for example, the at least a first biomarker maycomprise GFAP, UCH L1, and NF-L and may be exclusive of Tau.

In certain embodiments, for example, the method may distinguish betweenastrocytic injury and neuronal/axonal injury. In certain embodiments,for example, the at least a first biomarker may comprise GFAP. Incertain embodiments, for example, the at least a second biomarker maycomprise UCH L1 and/or NF-L. In certain embodiments, for example, themethod may distinguish between a neurological condition withintracranial hemorrhage and a neurological condition withoutintracranial hemorrhage. In certain embodiments, for example, the atleast a first biomarker may comprise NF-L.

In certain embodiments, for example, the method may distinguish betweena first type of MS and a second type of MS. In certain embodiments, forexample, the first type of MS and the second type of MS may be selectedfrom the group consisting of relapsing-remitting MS, primary progressiveMS, progressive relapsing MS, and secondary progressive MS. In certainembodiments, for example, the at least a first biomarker may compriseGFAP. In certain embodiments, for example, the at least a secondbiomarker may comprise UCH L1 or NF-L. In certain embodiments, forexample, the at least a second biomarker may be at least two biomarkers,the at least two biomarkers comprising UCH L1 and NF-L.

B. In certain embodiments, for example, the at least one difference maybe classified using a classification model (for example a statisticalmodel such as a train statistical model). In certain embodiments, forexample, the classification model may be a function of (or utilizes asinputs) concentrations for at least one biomarker in the liquid sampleand in the group of healthy donor, the at least one biomarker selectedfrom the group consisting of NF-L, GFAP, UCH L1, and Tau.

Certain embodiments may provide, for example, a test for a neurologicalcondition (for example a neural injury, defect, disorder, or disease).In certain embodiments, for example, the test may comprise providing asample of venous blood plasma or serum from a subject. In certainembodiments, for example, the test may comprise diluting the sample witha diluent to form a liquid sample, the diluent comprising apredetermined concentration of at least one heterophilic interferenceinhibitor for at least one biomarker, the at least one biomarkerselected from the group consisting of NF-L, GFAP, UCH L1, and Tau. Incertain embodiments, for example, the test may comprise obtaining, viadigital immunoassay, signal readings for NF-L, GFAP, UCH L1, and Tau inthe liquid sample. In certain embodiments, for example, the test maycomprise computing concentrations for NF-L, GFAP, UCH L1, and Tau in theliquid sample based on a standard curve, the standard curve derived froma plurality of calibration solutions, the calibration solutionsexclusive of the heterophilic interference inhibitor.

A. In certain embodiments, for example, the at least one heterophilicinterference inhibitor may be configured to increase at least onedetectable signal of the immunoassay. In certain embodiments, forexample, one of the at least one detectable signal may be associatedwith NF-L. In certain embodiments, for example, the at least oneheterophilic interference inhibitor may comprise an immunoglobulin. Incertain embodiments, for example, the immunoglobulin may be a human (orhumanized) immunoglobulin. In certain embodiments, for example, theimmunoglobulin may be an IgG. In certain embodiments, for example, theimmunoglobulin may be a human IgG. In certain embodiments, for example,the at least one heterophilic interference inhibitor may be exclusive ofnon-human immunoglobulin.

B. In certain embodiments, for example, the sample diluent may comprisephosphate, NaCl, KCl, bovine serum albumin (BSA), MgCl₂, dextrose,bovine gamma globulin (BgG), urea, the non-ionic surfactant sold underthe trademark Triton™ X-100, the immunoassay blocker sold under thetrademark TRU Block™, the heterophile blocking agent sold under thetrademark Superchemiblock™, human IgG, the preservative sold under thetrademark ProClin™ 300, or a combination of two or more of theforegoing. In certain embodiments, for example, the sample diluentfurther may comprise 50 mM phosphate, 137 mM NaCl, 2.7 mM KCl, 0.02%BSA, 1 mM MgCl₂, 0.06% dextrose, 0.01% BgG, 5 mM urea, 0.5% Triton™X-100, 10 mcg/mL TRU Block™, 50 mcg/mL Superchemiblock™, 5 mg/mL humanIgG, and 0.05% ProClin™ 300.

C. In certain embodiments, for example, the standard curve may be usedto compute a spike recovery for at least one of NF-L, GFAP, Tau, and UCHL1 (for example at least two of NF-L, GFAP, Tau, and UCH L1, at leastthree of NF-L, GFAP, Tau, and UCH L1, or each of NF-L, GFAP, Tau, andUCH L1) of between 80% and 120% (for example of between 95% and 105%) inthe liquid sample (for example when the liquid sample is spiked withNF-L at a concentration of between 5 pg/mL and 1000 pg/mL, for examplebetween 5 pg/mL and 100 pg/mL, between 5 pg/mL and 50 pg/mL, between 5pg/mL and 10 pg/mL, between 10 pg/mL and 100 pg/mL, between 10 pg/mL and50 pg/mL, 5 pg/mL 5 pg/mL, 10 pg/mL, 50 pg/mL, 100 pg/mL, or 1000 pg/mL;for example when the liquid sample is spiked with NF-L and GFAP atconcentrations of between 5 pg/mL and 1000 pg/mL, for example between 5pg/mL and 100 pg/mL, between 5 pg/mL and 50 pg/mL, between 5 pg/mL and10 pg/mL, between 10 pg/mL and 100 pg/mL, between 10 pg/mL and 50 pg/mL,5 pg/mL 5 pg/mL, 10 pg/mL, 50 pg/mL, 100 pg/mL, or 1000 pg/mL; forexample when the liquid sample is spiked with NF-L, GFAP, and Tau atconcentrations of between 5 pg/mL and 1000 pg/mL, for example between 5pg/mL and 100 pg/mL, between 5 pg/mL and 50 pg/mL, between 5 pg/mL and10 pg/mL, between 10 pg/mL and 100 pg/mL, between 10 pg/mL and 50 pg/mL,5 pg/mL 5 pg/mL, 10 pg/mL, 50 pg/mL, 100 pg/mL, or 1000 pg/mL; forexample when the liquid sample is spiked with the at least one of NF-L,GFAP, Tau, and UCH L1 at a concentrations of between 5 pg/mL and 1000pg/mL, for example between 5 pg/mL and 100 pg/mL, between 5 pg/mL and 50pg/mL, between 5 pg/mL and 10 pg/mL, between 10 pg/mL and 100 pg/mL,between 10 pg/mL and 50 pg/mL, 5 pg/mL 5 pg/mL, 10 pg/mL, 50 pg/mL, 100pg/mL, or 1000 pg/mL; or for example when the liquid sample is spikedwith the at least one of NF-L, GFAP, Tau, and UCH L1 at a concentrationfor each of the at least at least one of NF-L, GFAP, Tau, and UCH L1 ofbetween 5 pg/mL and 1000 pg/mL, for example between 5 pg/mL and 100pg/mL, between 5 pg/mL and 50 pg/mL, between 5 pg/mL and 10 pg/mL,between 10 pg/mL and 100 pg/mL, between 10 pg/mL and 50 pg/mL, 5 pg/mL 5pg/mL, 10 pg/mL, 50 pg/mL, 100 pg/mL, or 1000 pg/mL). In certainembodiments, for example, the standard curve may be used to compute aseries of concentrations for at least one of NF-L, GFAP, Tau, and UCH L1(for example at least two of NF-L, GFAP, Tau, and UCH L1, at least threeof NF-L, GFAP, Tau, and UCH L1, or each of NF-L, GFAP, Tau, and UCH L1)that are between 80% and 140% (for example between 80% and 125% orbetween 90% and 115%) proportional to one another when the liquid sample(for example a serum sample, plasma sample, or CSF sample) is diluted bybetween 2 times and 128 times (for example between 4 times and 64 times,such as 4 times, 8 times, 16 times, 32 times, and 64 times diluted) bythe sample diluent.

Certain embodiments may provide, for example, an assay indicated by atraumatic event. In certain embodiments, for example, the assay maycomprise providing a first liquid sample, the first liquid samplederived from first physiological fluid taken from a subject within 24hours of the event. In certain embodiments, for example, the assay maycomprise exposing at least a portion of the liquid sample to a pluralityof capture objects, the plurality of capture objects comprising bindingsurfaces having affinity for a plurality of biomarkers, the plurality ofbiomarkers comprising: NF-L, GFAP, UCH L1, and Tau. In certainembodiments, for example, the assay may comprise binding at least onecapture object of the plurality of capture objects to at least onebiomarker of the plurality of biomarkers. In certain embodiments, forexample, the assay may comprise verifying that a statisticallysignificant proportion of the exposed plurality of capture objects arenot bound to any of the at least two biomarkers. In certain embodiments,for example, the assay may comprise quantifying first concentrations ofthe plurality of biomarkers. In certain embodiments, for example, theassay may comprise applying a statistical test to the firstconcentrations at a p-value of less than 0.05 (for example less than0.01, 0.001, or 0.0001) to assess a risk of a neurological condition(for example a TBI).

A. In certain embodiments, for example, the statistical test may utilizebiomarker concentrations obtained for a group of healthy donors asinputs. In certain embodiments, for example, the statistical test mayutilize second biomarker concentrations, the second biomarkerconcentrations quantified from a second liquid sample, the second liquidsample derived from second physiological fluid taken from the subject.

B. In certain embodiments, for example, the second physiological fluidmay be taken from the subject at a different time from the time whichthe first physiological fluid is taken.

C. In certain embodiments, for example, at least one concentration ofthe first concentrations may be indicative of the neurological conditionat a level of less than 1 pmol/L. In certain embodiments, for example,the event may be child birth resulting in a neonate. In certainembodiments, for example, the neonate may be at risk of hypoxia duringchild birth.

Certain embodiments may provide, for example, a method to detect aneurological condition in a subject. In certain embodiments, forexample, the method may comprise performing the assay of indicated by atraumatic event, comprising: i) providing a first liquid sample, thefirst liquid sample derived from first physiological fluid taken from asubject within 24 hours of the event; ii) exposing at least a portion ofthe liquid sample to a plurality of capture objects, the plurality ofcapture objects comprising binding surfaces having affinity for aplurality of biomarkers, the plurality of biomarkers comprising: NF-L,GFAP, UCH L1, and Tau; iii) binding at least one capture object of theplurality of capture objects to at least one biomarker of the pluralityof biomarkers; iv) verifying that a statistically significant proportionof the exposed plurality of capture objects are not bound to any of theat least two biomarkers; and v) quantifying first concentrations of theplurality of biomarkers; and vi) applying a statistical test to thefirst concentrations at a p-value of less than 0.05 to assess a risk ofthe neurological condition (for example a TBI or MS). In certainembodiments, for example, the method may comprise calculating at leastone classification value based on a classification model of the firstconcentrations. In certain embodiments, for example, the method maycomprise assigning a risk of the neurological condition, comprising:comparing the at least one classification value to at least onethreshold value.

Certain embodiments may provide, for example, a method for detecting aneurological condition (for example a TBI or MS). In certainembodiments, for example, the method may comprise providing a liquidsample derived from a sample of physiological fluid taken from asubject. In certain embodiments, for example, the method may comprisediluting the liquid sample to adjust the liquid sample to within aworking range in a digital immunoassay, the working range comprising: a)a plurality of biomarkers comprising NF-L, GFAP, UCH L1, and Tau, atleast one biomarker of the plurality of biomarkers present at aconcentration that is greater than a corresponding at least one limit ofquantification of the digital immunoassay; and b) at least one thresholdindicative of the neurological condition that is greater than the atleast one corresponding limit of quantification. In certain embodiments,for example, the method may comprise quantifying concentrations NF-L,GFAP, UCH L1, and Tau via the digital immunoassay. In certainembodiments, for example, the digital immunoassay may be a multipleximmunoassay for NF-L, GFAP, UCH L1, and Tau.

Certain embodiments may provide, for example, a method for detecting aneurological condition. In certain embodiments, for example, the methodmay comprise providing a liquid sample derived from a sample ofphysiological fluid taken from a subject. In certain embodiments, forexample, the method may comprise diluting a portion of the liquid sampleto align concentrations of at least two biomarkers with a classificationmodel for determining a risk of the neurological condition, the at leasttwo biomarkers selected from the group consisting of NF-L, GFAP, UCH L1,and Tau. In certain embodiments, for example, the method may comprisequantifying the at least two biomarkers concentrations via a digitalimmunoassay for NF-L, GFAP, UCH L1, and Tau. In certain embodiments, forexample, the classification model may be calibrated to a standard curve.

Certain embodiments may provide, for example, a dual-test method todetect a neurological condition. In certain embodiments, for example,the dual-test method may comprise a first assessment for theneurological condition in a subject. In certain embodiments, forexample, the dual-test method may comprise performing, in response tothe first assessment, a second assessment for the neurologicalcondition, which second assessment is a multiplex immunoassay for NF-L,GFAP, UCH L1, and Tau on a fluid sample derived from the subject. Incertain embodiments, for example, the first assessment may comprise a CTscan. In certain embodiments, for example, the first assessment maycomprise an MRI scan.

Certain embodiments may provide, for example, a method to screen for aneurological condition. In certain embodiments, for example, the methodmay comprise providing a liquid sample derived from a sample ofphysiological fluid taken from a subject. In certain embodiments, forexample, the method may comprise quantifying a first component in afirst portion of the liquid sample. In certain embodiments, for example,the method may comprise computing a dilution factor based on thequantified first component. In certain embodiments, for example, themethod may comprise diluting a second portion of the liquid sample bythe dilution factor. In certain embodiments, for example, the method maycomprise quantifying NF-L, GFAP, UCH L1, and Tau in the second portionof the liquid sample. In certain embodiments, for example, the firstcomponent concentration may be insensitive to changes in central nervoussystem (CNS) function associated with onset of one or more neurologicalconditions.

Certain embodiments may provide, for example, a method to detect aneurological condition. In certain embodiments, for example, the methodmay comprise: diluting a sample of physiological fluid in a diluent toform a diluted sample. In certain embodiments, for example, the methodmay comprise: performing a multiplex immunoassay on the diluted sampleto obtain a plurality of measured parameters. In certain embodiments,for example, the method may comprise: obtaining concentration values forNF-L, GFAP, UCH L1, and Tau, comprising: inputting at least four of theplurality of measured parameters into a multivariate calibration model.

A. In certain embodiments, for example, the diluent may comprise humanIgG.

B. In certain embodiments, for example, the multivariate calibrationmodel may be derived from (for example may be fitted to) results of aseries of multiplex calibration immunoassays. In certain embodiments,for example, the series of multiplex calibration immunoassays maycomprise: i) a first calibration assay performed on a first calibrationsolution, the first calibration solution comprising NF-L at a first NF-Lconcentration, GFAP at a first GFAP concentration, UCH L1 at a first UCHL1 concentration, and Tau at a first Tau concentration; and ii) at leasta second calibration assay performed on an at least second calibrationsolution, the at least second calibration solution comprising NF-L at anat least second NF-L concentration, GFAP at an at least second GFAPconcentration, UCH L1 at an at least second UCH L1 concentration, andTau at an at least second Tau concentration. In certain embodiments, forexample, the series of multiplex immunoassays may comprise a calibrationassay on a calibration solution that is exclusive of NF-L, GFAP, UCH L1,and Tau.

Certain embodiments may provide, for example, a kit. In certainembodiments, for example, the kit may comprise: a plurality of captureagents configured to separately bind to two or more (for example threeor more) types of analytes selected from the group consisting of NF-L,GFAP, UCH L1, and Tau. In certain embodiments, for example, the kit maycomprise: a plurality of detection agents configured to separately bindto the selected two or more types of analytes. In certain embodiments,for example, the kit may comprise: a sample diluent. In certainembodiments, for example, the kit may comprise: at least one calibrationsolution comprising at least first predetermined concentrations of theselected two or more types of analytes.

A. In certain embodiments, for example, at least one of the plurality ofcapture agents may comprise a bead (for example a paramagnetic beadconfigured for use in a multiplex digital immunoassay). In certainembodiments, for example, at least one of the plurality of captureagents may comprise a tag. In certain embodiments, for example, the kitmay further comprise at least one bead, the at least one bead configuredto selectively bind to the tag.

B. In certain embodiments, for example, the sample diluent may comprisehuman IgG.

C. In certain embodiments, for example, the at least one calibrationsolution may be a concentrate. In certain embodiments, for example, theat least one calibration solution may be pre-diluted to a workingconcentration for one or more analytes.

Certain embodiments may provide, for example, a kit. In certainembodiments, for example, the kit may comprise: a plurality of captureagents configured to separately bind to two or more (for example threeor more) types of analytes selected from the group consisting of NF-L,GFAP, UCH L1, and Tau. In certain embodiments, for example, the kit maycomprise: a plurality of detection agents configured to separately bindto the selected two or more types of analytes. In certain embodiments,for example, the kit may comprise: a sample diluent comprising humanIgG. In certain embodiments, for example, the kit may comprise: aplurality of calibration solutions comprising a plurality ofpredetermined concentrations of the selected two or more types ofanalytes.

A. In certain embodiments, for example, the human IgG may be present inthe sample diluent at a concentration of between 0.1 mg/mL and 25 mg/mL,for example between 0.25 mg/mL and 15 mg/mL, between 0.25 mg/mL and 1.0mg/mL, between 1 mg/mL and 10 mg/mL, between 1 mg/mL and 3 mg/mL,between 3 mg/mL and 5 mg/mL, between 5 mg/mL and 7 mg/mL, between 7mg/mL and 9 mg/mL, between 9 mg/mL and 11 mg/mL, between 11 mg/mL and 13mg/mL, or the human IgG may be present in the diluent at a concentrationof between 13 mg/mL and 15 mg/mL. In certain embodiments, for example,the human IgG may be present in the sample diluent at a concentration of5 mg/mL. In certain embodiments, for example, the human IgG may bepresent in the sample diluent at a concentration of between 0.1 mcg/mLand 25 mcg/mL, for example between 0.25 mcg/mL and 15 mcg/mL, between0.25 mcg/mL and 1.0 mcg/mL, between 1 mcg/mL and 10 mcg/mL, between 1mcg/mL and 3 mcg/mL, between 3 mcg/mL and 5 mcg/mL, between 5 mcg/mL and7 mcg/mL, between 7 mcg/mL and 9 mcg/mL, between 9 mcg/mL and 11 mcg/mL,between 11 mcg/mL and 13 mcg/mL, or the human IgG may be present in thediluent at a concentration of between 13 mcg/mL and 15 mcg/mL. Incertain embodiments, for example, the human IgG may be present in thesample diluent at a concentration of 5 mcg/mL. In certain embodiments,for example, the sample diluent may further comprise at least oneheterophile blocking agent exclusive of the human IgG. In certainembodiments, for example, the at least one heterophile blocking agentmay be present in the sample diluent at a concentration of at least 1mcg/mL, for example at least 5 mcg/mL, at least 10 mcg/mL, at least 15mcg/mL, at least 20 mcg/mL, at least 25 mcg/mL or the at least oneheterophile blocking agent may be present in the sample diluent at aconcentration of at least 50 mcg/mL. In certain embodiments, forexample, the at least one heterophile blocking agent exclusive of humanIgG may be present in the sample diluent at a concentration (i.e., thetotal concentration of all heterophile blocking agents exclusive ofhuman IgG) of between 1 mcg/mL and 100 mcg/mL, for example between 5mcg/mL and 50 mcg/mL, between 10 mcg/mL and 20 mcg/mL, or the at leastone heterophile blocking agent may be present in the sample diluent at aconcentration of between 12 mcg/mL and 18 mcg/mL. In certainembodiments, for example, the human IgG may be present in the samplediluent at a concentration of between 1 mg/mL and 10 mg/mL and the atleast one heterophile blocking agent exclusive of human IgG may bepresent at a concentration of at least 10 mcg/mL (for example at least15 mcg/mL). In certain embodiments, for example, the at least oneheterophile blocking agent exclusive of human IgG may be present in thesample diluent at a concentration of 15 mcg/mL. In certain embodiments,for example, the at least one heterophile blocking agent may compriseTRU Block™. In certain embodiments, for example, the TRU Block™ may bepresent in the sample diluent at a concentration of between 1 mcg/mL and100 mcg/mL, for example between 2 mcg/mL and 25 mcg/mL, between 5 mcg/mLand 15 mcg/mL, or the TRU Block™ may be present in the sample diluent ata concentration of between 8 mcg/mL and 12 mcg/mL. In certainembodiments, for example, the TRU Block™ may be present in the samplediluent at a concentration of 10 mcg/mL. In certain embodiments, forexample, the at least one heterophile blocking agent may compriseSuperchemiblock™. In certain embodiments, for example, theSuperchemiblock™ may be present in the sample diluent at a concentrationof between 0.5 mcg/mL and 25 mcg/mL, for example between 1 mcg/mL and 15mcg/mL, between 2 mcg/mL and 10 mcg/mL, or the Superchemiblock™ may bepresent in the sample diluent at a concentration of between 3 mcg/mL and7 mcg/mL. In certain embodiments, for example, the Superchemiblock™ maybe present in the sample diluent at a concentration of 5 mcg/mL. Incertain embodiments, for example, the sample diluent may comprise humanIgG at a concentration of between 1 mg/mL and 10 mg/mL and at least oneheterophile blocking agent at a concentration of between 5 and 25mcg/mL, for example human IgG at a concentration of between 3 mg/mL and7 mg/mL and at least one heterophile blocking agent at a concentrationof between 10 and 20 mcg/mL, human IgG at a concentration of between 3mg/mL and 7 mg/mL and at least one heterophile blocking agent at aconcentration of between 13 and 17 mcg/mL, or human IgG at aconcentration of 5 mg/mL and at least one heterophile blocking agent ata concentration of 15 mcg/mL. In certain embodiments, for example, thesample diluent may comprise human IgG at a concentration of between 1mg/mL and 10 mg/mL, TRU Block™ at a concentration of 5 mcg/mL and 15mcg/mL, and Superchemiblock™ at a concentration of 1 mcg/mL and 10mcg/mL. In certain embodiments, for example, the sample diluent maycomprise human IgG at a concentration of between 3 mg/mL and 7 mg/mL,TRU Block™ at a concentration of 5 mcg/mL and 15 mcg/mL, andSuperchemiblock™ at a concentration of 1 mcg/mL and 10 mcg/mL. Incertain embodiments, for example, the sample diluent may comprise humanIgG at a concentration of between 1 mg/mL and 10 mg/mL, TRU Block™ at aconcentration of 7 mcg/mL and 12 mcg/mL, and Superchemiblock™ at aconcentration of 1 mcg/mL and 10 mcg/mL. In certain embodiments, forexample, the sample diluent may comprise human IgG at a concentration ofbetween 1 mg/mL and 10 mg/mL, TRU Block™ at a concentration of 5 mcg/mLand 15 mcg/mL, and Superchemiblock™ at a concentration of 3 mcg/mL and 7mcg/mL. In certain embodiments, for example, the sample diluent maycomprise human IgG at a concentration of 5 mg/mL, TRU Block™ at aconcentration of 10 mcg/mL, and Superchemiblock™ at a concentration of 5mcg/mL. In certain embodiments, for example, the plurality ofcalibration solutions may be pre-diluted for use to determine acalibration standard curve without further dilution.

B. In certain embodiments, for example, the two or more types ofanalytes may be four types of analytes consisting of NF-L, GFAP, UCH L1,and Tau. In certain embodiments, for example, the plurality ofcalibration solutions may be between 6 and 10 calibration solutions,inclusive of an NF-L-free, GFAP-free, UCH L1-free, and Tau-free controlsolution. In certain embodiments, for example, the plurality ofcalibration solutions may comprise: i) a first calibration solutioncomprising NF-L at a concentration of at least 0.5 pg/mL, GFAP at aconcentration of at least 1 pg/mL, UCH L1 at a concentration of at least10 pg/mL, and Tau at a concentration of at least 0.1 pg/mL; ii) a secondcalibration solution comprising NF-L at a concentration of at least 450pg/mL, GFAP at a concentration of at least 900 pg/mL, UCH L1 at aconcentration of at least 9000 pg/mL, and Tau at a concentration of atleast 90 pg/mL; and iii) at least a third calibration solutioncomprising NF-L at a concentration of between 10 pg/mL and 450 pg/mL,GFAP at a concentration of between 20 pg/mL and 900 pg/mL, UCH L1 at aconcentration of between 200 pg/mL and 9000 pg/mL, and Tau at aconcentration of between 2 pg/mL and 90 pg/mL.

C. In certain embodiments, for example, at least one of the plurality ofcapture agents may comprise a paramagnetic bead configured for use in amultiplex digital immunoassay analyzer. In certain embodiments, forexample, the plurality of detection agents may comprise: i) an NF-Ldetection agent configured to bind to NF-L; ii) a GFAP detection agentconfigured to bind to GFAP; iii) a UCH L1 detection agent configured tobind to UCH L1; and iv) a Tau detection agent configured to bind to Tau.

Certain embodiments may provide, for example, a method to detect aneurological condition. In certain embodiments, for example, the methodmay comprise: diluting a sample of physiological fluid in a diluent toform a diluted sample. In certain embodiments, for example, the methodmay comprise: performing a multiplex immunoassay on the diluted sampleto obtain a plurality of measured parameters. In certain embodiments,for example, the method may comprise: obtaining concentration values forNF-L, GFAP, UCH L1, and Tau, comprising: inputting at least four of theplurality of measured parameters into a multivariate calibration model.

A. In certain embodiments, for example, the measured parameters maycomprise signal readings from a multiplex spotted well immunoassay. Incertain embodiments, for example, the measured parameters may be derivedfrom Poisson and/or Gaussian distribution analysis of results of adigital immunoassay.

B. In certain embodiments, for example, the multivariate calibrationmodel may be derived from a results of a series of multiplex calibrationimmunoassays. In certain embodiments, for example, the multivariatecalibration model may be derived from one or more polynomial regressionsusing the results as inputs. In certain embodiments, for example, themultivariate calibration model may have R² values of at least 0.95 fortwo or more of: i) NF-L at a concentration of between 1 pg/mL and 50pg/mL; ii) Tau at a concentration of between 0.1 pg/mL and 8 pg/mL; iii)GFAP at a concentration of between 5 pg/mL and 100 pg/mL; and iv) UCH L1at a concentration of between 20 pg/mL and 500 pg/mL. In certainembodiments, for example, the multivariate calibration model may have R²values of at least 0.95 for two or more of: i) NF-L at a concentrationof between 0.1 pg/mL and 10 pg/mL; ii) Tau at a concentration of between0.1 pg/mL and 8 pg/mL; iii) GFAP at a concentration of between 10 pg/mLand 500 pg/mL; and iv) UCH L1 at a concentration of between 20 pg/mL and100 pg/mL.

C. In certain embodiments, for example, the series of multiplexcalibration immunoassays may comprise: i) a first calibration assayperformed on a first calibration solution, the first calibrationsolution comprising NF-L at a first NF-L concentration, GFAP at a firstGFAP concentration, UCH L1 at a first UCH L1 concentration, and Tau at afirst Tau concentration; and ii) at least a second calibration assayperformed on an at least second calibration solution, the at leastsecond calibration solution comprising NF-L at an at least second NF-Lconcentration, GFAP at an at least second GFAP concentration, UCH L1 atan at least second UCH L1 concentration, and Tau at an at least secondTau concentration.

D. In certain embodiments, for example, a comparative series ofmultiplex immunoassays for NF-L, GFAP, UCH L1, and Tau may be performedto obtain a series of comparative measured parameters (for examplesignal readings or digital assay results) corresponding to at least oneof NF-L, GFAP, Tau, and UCH L1 (for example at least two of NF-L, GFAP,Tau, and UCH L1, at least three of NF-L, GFAP, Tau, and UCH L1, or eachof NF-L, GFAP, Tau, and UCH L1) that are between 80% and 140% (forexample between 80% and 125% or between 90% and 115%) proportional toone another when the sample of physiological fluid (for example a serumsample, plasma sample, or CSF sample) is diluted by between 2 times and128 times (for example between 4 times and 64 times, such as 4 times, 8times, 16 times, 32 times, and 64 times diluted) by the sample diluent.In certain embodiments, for example, the multivariate calibration modelmay be used to obtain a series of comparative concentration values forat least one of NF-L, GFAP, Tau, and UCH L1 (for example at least two ofNF-L, GFAP, Tau, and UCH L1, at least three of NF-L, GFAP, Tau, and UCHL1, or each of NF-L, GFAP, Tau, and UCH L1) that are between 80% and140% (for example between 80% and 125% or between 90% and 115%)proportional to one another when the sample of physiological fluid (forexample a serum sample, plasma sample, or CSF sample) is diluted bybetween 2 times and 128 times (for example between 4 times and 64 times,such as 4 times, 8 times, 16 times, 32 times, and 64 times diluted) bythe sample diluent. In certain embodiments, for example, the diluent maycomprise between 1 mg/mL and 50 mg/mL human IgG (for example between 1mg/mL and 10 mg/mL, between 3 mg/mL and 7 mg/mL, or 5 mg/mL human IgG).

Certain embodiments may provide, for example, a test for a neurologicalcondition. In certain embodiments, for example, the test may comprise:providing a liquid sample derived from a sample of physiological fluid.In certain embodiments, for example, the test may comprise: obtaining,via a multiplex immunoassay, concentrations of NF-L, GFAP, UCH L1, andTau in the liquid sample. In certain embodiments, for example, the testmay comprise: calculating at least one classification value based on amultivariate classification model using the concentrations as inputs tothe multivariate classification model. In certain embodiments, forexample, the test may comprise: assigning a risk of the neurologicalcondition, comprising: comparing the at least one classification valueto at least one threshold value.

A. In certain embodiments, for example, the multivariate classificationmodel may predict TBI with an ROC curve having an AUC of at least 0.85.In certain embodiments, for example, the multivariate classificationmodel may predict a TBI with a true positive rate of at least 75% at afalse positive rate of less than 25%.

B. In certain embodiments, for example, the test may further comprise:obtaining a CT scan result negative for the neurological condition priorto performing the multiplex immunoassay. In certain embodiments, forexample, the multivariate classification model may comprise a neuralnetwork.

Certain embodiments may provide, for example, a test for a neurologicalcondition, comprising: i) providing a liquid sample derived from asample of physiological fluid; ii) obtaining, via a single multipleximmunoassay, concentrations of NF-L, GFAP, UCH L1, and Tau in the liquidsample; iii) calculating at least one classification value based on amultivariate classification model using the concentrations as inputs tothe multivariate classification model; and iv) assigning a risk of theneurological condition, comprising: comparing the at least oneclassification value to at least one threshold value.

Certain embodiments may provide, for example, a single-sample test for aneurological condition, comprising: i) providing a liquid sample derivedfrom a single sample of physiological fluid from a subject; ii)obtaining, via at least one immunoassay, concentrations of NF-L, GFAP,UCH L1, and Tau in the liquid sample; and iii) assigning a risk of theneurological condition in the subject, comprising: determining at leastone measure of significance of differences between the concentrations ofNF-L, GFAP, UCH L1, and Tau in the liquid sample and concentrations ofNF-L, GFAP, UCH L1, and Tau in a group of healthy donors.

Certain embodiments may provide, for example, a protocol indicated by apotential neurological condition, comprising a CT scan positive for aneurological condition; followed by one of the single-sample tests forthe neurological condition disclosed herein.

Certain embodiments may provide, for example, a modified protocolindicated by a potential neurological condition, comprising a protocolfor detection of the neurological condition comprising a CT scan and inwhich an MRI scan is replaced by one of the single-sample tests for theneurological condition disclosed herein.

Certain embodiments may provide, for example, a single-assay test for aneurological condition, comprising: i) providing at least one liquidsample derived from a single sample of physiological fluid from asubject; ii) obtaining, via an immunoassay, concentrations of NF-L,GFAP, UCH L1, and Tau in the at least one liquid sample; and iii)assigning a risk of the neurological condition in the subject,comprising: determining at least one measure of significance ofdifferences between the concentrations of NF-L, GFAP, UCH L1, and Tau inthe liquid sample and concentrations of NF-L, GFAP, UCH L1, and Tau in agroup of healthy donors.

Certain embodiments may provide, for example, a dual-sample test for aneurological condition, comprising: i) providing, from a subject: a) afirst liquid sample derived from a sample of a first physiological fluidtaken at a first time; and b) a second liquid sample derived from asample of a second physiological fluid taken at a second time; ii)obtaining, via immunoassay, concentrations of NF-L, GFAP, UCH L1, andTau in the first liquid sample and in the second liquid sample; iii)assigning a risk of the neurological condition in the subject,comprising: determining at least one measure of significance ofdifferences between the concentrations of NF-L, GFAP, UCH L1, and Tau inthe first liquid sample and the concentrations of NF-L, GFAP, UCH L1,and Tau in the second liquid sample.

Certain embodiments may provide, for example, a dual-sample test for aneurological condition, comprising: i) providing, from a subject: a) afirst liquid sample derived from a first physiological fluid sampletaken at a first time; and b) at least a second liquid sample derivedfrom at least a second physiological fluid sample taken at least asecond time; ii) obtaining, via immunoassay, concentrations of NF-L,GFAP, UCH L1, and Tau in the first liquid sample and in the at least thesecond liquid sample; iii) assigning a risk of the neurologicalcondition in the subject, comprising: determining at least one measureof significance of differences between the concentrations of NF-L, GFAP,UCH L1, and Tau in the first liquid sample and the concentrations ofNF-L, GFAP, UCH L1, and Tau in the at least the second liquid sample.

Certain embodiments may provide, for example, a method to distinguishbetween types of neurological condition, comprising: i) providing aliquid sample derived from a sample of physiological fluid from asubject; ii) obtaining, via at least one immunoassay, concentrations ofNF-L, GFAP, UCH L1, and Tau in the liquid sample; iii) distinguishingbetween types of neurological condition, comprising: a) classifying asstatistically significant a difference between at least a firstbiomarker concentration of the concentrations of NF-L, GFAP, UCH L1, andTau in the liquid sample and concentrations of NF-L, GFAP, UCH L1, andTau in a group of healthy donors; and b) determining that a differencebetween at least a second biomarker concentration of the concentrationsof NF-L, GFAP, UCH L1, and Tau in the liquid sample and theconcentrations of NF-L, GFAP, UCH L1, and Tau in a group of healthydonors is statistically insignificant.

Certain embodiments may provide, for example, a test for a neurologicalcondition, comprising i) providing a sample of venous blood plasma orserum from a subject; ii) diluting the sample with a diluent to form aliquid sample, the diluent comprising a predetermined concentration ofat least one heterophilic interference inhibitor for at least onebiomarker selected from the group consisting of NF-L, GFAP, UCH L1, andTau; iii) obtaining, via digital immunoassay, signal readings for NF-L,GFAP, UCH L1, and Tau in the liquid sample; and iii) computingconcentrations for NF-L, GFAP, UCH L1, and Tau in the liquid samplebased on a standard curve, the standard curve derived from a pluralityof calibration solutions, the calibration solutions exclusive of theheterophilic interference inhibitor.

Certain embodiments may provide, for example, an assay indicated by atraumatic event, comprising: i) providing a first liquid sample, thefirst liquid sample derived from first physiological fluid taken from asubject within 24 hours of the event; ii) exposing at least a portion ofthe liquid sample to a plurality of capture objects, the plurality ofcapture objects comprising binding surfaces having affinity for aplurality of biomarkers, the plurality of biomarkers comprising: NF-L,GFAP, UCH L1, and Tau; iii) binding at least one capture object of theplurality of capture objects to at least one biomarker of the pluralityof biomarkers; iv) verifying that a statistically significant proportionof the exposed plurality of capture objects are not bound to any of theat least two biomarkers; v) quantifying first concentrations of theplurality of biomarkers; and vi) applying a statistical test to thefirst concentrations at a p-value of less than 0.05 to assess a risk ofa neurological condition.

Certain embodiments may provide, for example, a method to detect aneurological condition in a subject, comprising: i) performing an assayindicated by a traumatic event, comprising: a) providing a first liquidsample, the first liquid sample derived from first physiological fluidtaken from a subject within 24 hours of the event; b) exposing at leasta portion of the liquid sample to a plurality of capture objects, theplurality of capture objects comprising binding surfaces having affinityfor a plurality of biomarkers, the plurality of biomarkers comprising:NF-L, GFAP, UCH L1, and Tau; c) binding at least one capture object ofthe plurality of capture objects to at least one biomarker of theplurality of biomarkers; d) verifying that a statistically significantproportion of the exposed plurality of capture objects are not bound toany of the at least two biomarkers; and e) quantifying firstconcentrations of the plurality of biomarkers; and f) applying astatistical test to the first concentrations at a p-value of less than0.05 to assess a risk of a neurological condition; ii) calculating atleast one classification value based on a classification model of thefirst concentrations; and iii) assigning a risk of the neurologicalcondition, comprising: comparing the at least one classification valueto at least one threshold value.

Certain embodiments may provide, for example, a method for detecting aneurological condition, comprising: i) providing a liquid sample derivedfrom a sample of physiological fluid taken from a subject; ii) dilutingthe liquid sample to adjust the liquid sample to within a working rangein a digital immunoassay, the working range comprising: a) a pluralityof biomarkers comprising NF-L, GFAP, UCH L1, and Tau, at least onebiomarker of the plurality of biomarkers present at a concentration thatis greater than a corresponding at least one limit of quantification ofthe digital immunoassay; and b) at least one threshold indicative of theneurological condition that is greater than the at least onecorresponding limit of quantification; and iii) quantifyingconcentrations NF-L, GFAP, UCH L1, and Tau via the digital immunoassay.

Certain embodiments may provide, for example, a method for detecting aneurological condition, comprising: i) providing a liquid sample derivedfrom a sample of physiological fluid taken from a subject; ii) dilutinga portion of the liquid sample to align concentrations of at least twobiomarkers with a classification model for determining a risk of theneurological condition, the at least two biomarkers selected from thegroup consisting of NF-L, GFAP, UCH L1, and Tau; and iii) quantifyingthe at least two biomarkers concentrations via a digital immunoassay forNF-L, GFAP, UCH L1, and Tau.

Certain embodiments may provide, for example, a dual-test method todetect a neurological condition, comprising: i) a first assessment forthe neurological condition in a subject; and ii) performing, in responseto the first assessment, a second assessment for the neurologicalcondition, which second assessment is a multiplex immunoassay for NF-L,GFAP, UCH L1, and Tau on a fluid sample derived from the subject.

Certain embodiments may provide, for example, a method to screen for aneurological condition, comprising: i) providing a liquid sample derivedfrom a sample of physiological fluid taken from a subject; ii)quantifying a first component in a first portion of the liquid sample;iii) computing a dilution factor based on the quantified firstcomponent; iv) diluting a second portion of the liquid sample by thedilution factor; and v) quantifying NF-L, GFAP, UCH L1, and Tau in thesecond portion of the liquid sample.

Certain embodiments may provide, for example, a method to detect aneurological condition, comprising: i) diluting a sample ofphysiological fluid in a diluent to form a diluted sample; ii)performing a multiplex immunoassay on the diluted sample to obtain aplurality of measured parameters; and iii) obtaining concentrationvalues for NF-L, GFAP, UCH L1, and Tau, comprising: inputting at leastfour of the plurality of measured parameters into a multivariatecalibration model.

Certain embodiments may provide, for example, a kit, comprising: i) aplurality of capture agents configured to separately bind to two or more(for example three or more) types of analytes selected from the groupconsisting of NF-L, GFAP, UCH L1, and Tau; ii) a plurality of detectionagents configured to separately bind to the selected two or more typesof analytes; iii) a sample diluent; and iv) at least one calibrationsolution comprising at least first predetermined concentrations of theselected two or more types of analytes.

Certain embodiments may provide, for example, a kit, comprising: i) aplurality of capture agents configured to separately bind to two or more(for example three or more) types of analytes selected from the groupconsisting of NF-L, GFAP, UCH L1, and Tau; ii) a plurality of detectionagents configured to separately bind to the selected two or more typesof analytes; iii) a sample diluent comprising human IgG; and iv)plurality of calibration solutions comprising a plurality ofpredetermined concentrations of the selected two or more types ofanalytes.

Certain embodiments may provide, for example, a method to detect aneurological condition, comprising: i) diluting a sample ofphysiological fluid in a diluent to form a diluted sample; ii)performing a multiplex immunoassay on the diluted sample to obtain aplurality of measured parameters; and iii) obtaining concentrationvalues for NF-L, GFAP, UCH L1, and Tau, comprising: inputting at leastfour of the plurality of measured parameters into a multivariatecalibration model.

Certain embodiments may provide, for example, a test for a neurologicalcondition, comprising: i) providing a liquid sample derived from asample of physiological fluid; ii) obtaining, via a multipleximmunoassay, concentrations of NF-L, GFAP, UCH L1, and Tau in the liquidsample; iii) calculating at least one classification value based on amultivariate classification model using the concentrations as inputs tothe multivariate classification model; and iv) assigning a risk of theneurological condition, comprising: comparing the at least oneclassification value to at least one threshold value.

Certain embodiments may provide, for example, a kit, comprising: i) aplurality of capture agents configured to separately bind to two or moretypes of analytes selected from the group consisting of NF-L, GFAP, UCHL1, and Tau; ii) a plurality of detection agents configured toseparately bind to the selected two or more types of analytes; iii) asample diluent comprising human IgG at a concentration of between 1mg/mL and 10 mg/mL; and iv) a plurality of calibration solutionscomprising a plurality of predetermined concentrations of the selectedtwo or more types of analytes.

A. In certain embodiments, for example, the sample diluent further maycomprise at least 15 mcg/mL of at least one heterophile blocking agentexclusive of the human IgG. In certain embodiments, for example, theplurality of calibration solutions may be pre-diluted for use todetermine a calibration standard curve without further dilution. Incertain embodiments, for example, the two or more types of analytes maybe four types of analytes consisting of NF-L, GFAP, UCH L1, and Tau. Incertain embodiments, for example, the plurality of calibration solutionsmay be between 6 and 10 calibration solutions, inclusive of anNF-L-free, GFAP-free, UCH L1-free, and Tau-free control solution. Incertain embodiments, for example, the plurality of calibration solutionsmay comprise: i) a first calibration solution comprising NF-L at aconcentration of at least 0.5 pg/mL, GFAP at a concentration of at least1 pg/mL, UCH L1 at a concentration of at least 10 pg/mL, and Tau at aconcentration of at least 0.1 pg/mL; ii) a second calibration solutioncomprising NF-L at a concentration of at least 450 pg/mL, GFAP at aconcentration of at least 900 pg/mL, UCH L1 at a concentration of atleast 9000 pg/mL, and Tau at a concentration of at least 90 pg/mL; andiii) at least a third calibration solution comprising NF-L at aconcentration of between 10 pg/mL and 450 pg/mL, GFAP at a concentrationof between 20 pg/mL and 900 pg/mL, UCH L1 at a concentration of between200 pg/mL and 9000 pg/mL, and Tau at a concentration of between 2 pg/mLand 90 pg/mL. In certain embodiments, for example, at least one of theplurality of capture agents may comprise a paramagnetic bead configuredfor use in a multiplex digital immunoassay analyzer. In certainembodiments, for example, the plurality of detection agents maycomprise: i) an NF-L detection agent configured to bind to NF-L; ii) aGFAP detection agent configured to bind to GFAP; iii) a UCH L1 detectionagent configured to bind to UCH L1; and iv) a Tau detection agentconfigured to bind to Tau.

Certain embodiments may provide, for example, a method to detect aneurological condition, comprising: i) diluting a sample ofphysiological fluid in a diluent to form a diluted sample; ii)performing a multiplex immunoassay on the diluted sample to obtain aplurality of at least four measured parameters; and iii) obtainingconcentration values for NF-L, GFAP, UCH L1, and Tau, comprising:inputting at least four of the plurality of at least four measuredparameters into a multivariate calibration model.

A. In certain embodiments, for example, the plurality of the at leastfour measured parameters may comprise signal readings from a multiplexspotted well immunoassay. In certain embodiments, for example, theplurality of the at least four measured parameters may be derived fromPoisson and/or Gaussian distribution analysis of results of a multiplexdigital immunoassay. In certain embodiments, for example, themultivariate calibration model may be derived from results of a seriesof multiplex calibration immunoassays. In certain embodiments, forexample, the multivariate calibration model may provide a series ofcomparative concentration values for at least at least two of NF-L,GFAP, Tau, and UCH L1 that are between 80% and 140% proportional whenthe sample of physiological fluid is diluted between 4 times and 64times. In certain embodiments, for example, the multivariate calibrationmodel may have R² values of at least 0.95 for two or more of: i) NF-L ata concentration of between 1 pg/mL and 50 pg/mL; ii) Tau at aconcentration of between 0.1 pg/mL and 8 pg/mL; iii) GFAP at aconcentration of between 5 pg/mL and 100 pg/mL; and iv) UCH L1 at aconcentration of between 20 pg/mL and 500 pg/mL. In certain embodiments,for example, the series of multiplex calibration immunoassays maycomprise: i) a first calibration assay performed on a first calibrationsolution, the first calibration solution comprising NF-L at a first NF-Lconcentration, GFAP at a first GFAP concentration, UCH L1 at a first UCHL1 concentration, and Tau at a first Tau concentration; and ii) at leasta second calibration assay performed on an at least second calibrationsolution, the at least second calibration solution comprising NF-L at anat least second NF-L concentration, GFAP at an at least second GFAPconcentration, UCH L1 at an at least second UCH L1 concentration, andTau at an at least second Tau concentration.

Certain embodiments may provide, for example, a test for a neurologicalcondition, comprising: i) providing a liquid sample derived from asample of physiological fluid; ii) obtaining, via a multipleximmunoassay, concentrations of NF-L, GFAP, UCH L1, and Tau in the liquidsample; iii) calculating at least one classification value based on amultivariate classification model using the concentrations as inputs tothe multivariate classification model; and iv) assigning a risk of theneurological condition, comprising: comparing the at least oneclassification value to at least one threshold value.

A. In certain embodiments, for example, the multivariate classificationmodel may predict a traumatic brain injury with an ROC curve having anAUC of at least 0.85. In certain embodiments, for example, themultivariate classification model may predict a traumatic brain injurywith a true positive rate of at least 75% at a false positive rate ofless than 25%. In certain embodiments, for example, the test may furthercomprise: obtaining a CT scan result negative for the neurologicalcondition prior to performing the multiplex immunoassay. In certainembodiments, for example, the multivariate classification model maycomprise a neural network.

Methods and kits for determining a measure of the concentration of apanel neurological biomarkers in a sample derived from a patient aregenerally provided.

The subject matter of the present invention involves, in some cases,interrelated products, alternative solutions to a particular problem,and/or a plurality of different uses of one or more systems and/orarticles.

In certain embodiments, for example (for example in one set ofembodiments), the methods, tests, assays, kits, or systems (for examplemethods) may comprise determining a measure of the concentration of apanel of biomarkers in a sample, comprising performing an assay on asample derived from a human and determining a measure of theconcentration of NF-L and at least one other biomarker selected from thegroup consisting of GFAP, UCH L1, and Tau, in the sample collected fromthe patient. Certain embodiments, for example, may provide an assayhaving an LOQ of no greater than 0.1 pg/mL for Tau protein, no greaterthan 1 pg/mL for NF-L and GFAP, and no greater than 5 pg/mL for UCH L1.

In certain embodiments, for example (for example in another set ofembodiments), the methods, tests, assays, kits, or systems (for examplekits) may be used for determining a measure of the concentration of apanel of biomarkers in a sample comprises a plurality of captureobjects, each having a binding surface comprising a plurality of capturecomponents, a plurality of a first type of binding ligand havingaffinity for NF-L, and a plurality of a second type of binding ligandand a third type of binding ligand having affinity for at least twoother biomarkers selected from the group consisting of GFAP, UCH L1, andTau.

Other advantages and novel features of the present invention will becomeapparent from the following detailed description of various non-limitingembodiments of the invention when considered in conjunction with theaccompanying figures. In cases where the present specification and adocument incorporated by reference include conflicting and/orinconsistent disclosure, the present specification shall control. If twoor more documents incorporated by reference include conflicting and/orinconsistent disclosure with respect to each other, then the documenthaving the later effective date shall control.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic depiction of a multiplex digital assay.

FIG. 2 is a schematic depiction of a multiplex spotted well assay.

FIG. 3A is a schematic flow diagram depicting a detection method,according to certain embodiments.

FIG. 3B is a schematic flow diagram depicting a detection method,according to certain embodiments.

FIG. 4A is a NF-L immunoassay dose-response curve, according to certainembodiments.

FIG. 4B is NF-L concentration in plasma, matched serum, and CSF fromhealthy donors.

FIG. 5A is a Tau protein immunoassay dose-response curve.

FIG. 5B is Tau protein concentration in plasma, matched serum, and CSFfrom healthy donors.

FIG. 6A is a GFAP immunoassay dose-response curve.

FIG. 6B is GFAP concentration in plasma, matched serum, and CSF fromhealthy donors.

FIG. 7A is a UCH L1 immunoassay dose-response curve.

FIG. 7B is UCH L1 concentration in plasma, matched serum, and CSF fromhealthy donors.

FIG. 8 is a diagram of GFAP concentration.

FIG. 9 is a diagram of NF-L concentration.

FIG. 10 is a diagram of GFAP concentration.

FIG. 11 is a diagram of NF-L concentration.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure is based, generally, on the discovery thatbiomarkers collateral to neural function can be rapidly quantified fromphysiological fluids at very low concentrations (for example femtomolar)and used to help characterize the neural health of a subject, inclusiveof detecting a neural condition, excluding a neural condition,predicting onset of a neural condition, and distinguishing between twoor more neurological conditions that present similarly. The presentdisclosure is further specifically based, in part, on the discovery ofmulti-component calibrators and sample diluent formulations andpreparation methods that improve the precision of biomarkermeasurements. Moreover, these formulations and methods can be applied toa samples obtained from a plurality of individuals (for example acombination of healthy individuals and others suffering from one or moreneurological conditions) to obtain data sets from which improvedclassification models are obtained. Kits based on the formulations andmethods disclosed herein enable a subject to benefit from these improvedclassification models.

Certain embodiments may provide, for example, methods, tests, protocol,assays, kits, and/or systems for detecting, diagnosing, distinguishing,and/or excluding a neurological condition. In certain embodiments, forexample, the neurological condition may comprise a disease. In certainembodiments, for example, the disease may be selected from the groupconsisting of Alzheimer's disease, motor neuron disease, frontotemporaldementia, HIV-associated dementia, progressive supranuclear palsy,Parkinson disease, Huntington's disease, Lewy Body dementia, dementiapugilistica, Creutzfeldt-Jakob disease, amyotrophic lateral sclerosis,transverse myelitis, MS, demyelination occurring after trauma to thebrain or spinal cord, or a combination of two or more of the foregoingdiseases.

In certain embodiments, for example, the neurological condition maycomprise a condition resulting from an injury (for example TBI). Incertain embodiments, for example, the condition resulting from an injurymay be selected from the group consisting of acute brain injury, spinalcord injury, peripheral nerve injury, ischaemic brain injury, TBI (orhead trauma), traumatic spinal cord injury (or spinal cord trauma),stroke related injury, concussion (optionally including post-concussionsyndrome), cerebral aneurism related injury, injury from general anoxia,hypoxia, hypoglycemia, hypotension, damage to retinal ganglion cells, aspinal cord injury (optionally including monoplegia, diplegia,paraplegia, hemiplegia and quadriplegia, or a combination of two or moreof the foregoing), demyelination occurring after trauma to the brain orspinal cord, brain injuries secondary to seizures (for example seizuresinduced by radiation, exposure to ionizing or iron plasma, nerve agents,cyanide, toxic concentrations of oxygen, neurotoxicity due to CNSmalaria or treatment with anti-malaria agents, trypanosomes, malarialpathogens, other CNS traumas, or a combination of two or more of theforegoing), injuries caused by procedures (for example proceduresresulting from embole, hyperfusion, or hypoxia), or a combination of twoor more of the foregoing conditions.

In certain embodiments, for example, the neurological condition maycomprise a defect. In certain embodiments, for example, the defect maybe selected from the group consisting of defects caused by defectivetissues or cells of the nervous system, defects caused by defectivetissues or cells that affect the nervous system (such as defective spinemorphogenesis and defects in dendritic spine morphology), or acombination of two or more of the foregoing defects.

In certain embodiments, for example, the neurological condition maycomprise a disorder. In certain embodiments, for example, the disordermay be selected from the group consisting of transverse myelitis, MS,demyelination occurring after trauma to the brain or spinal cord, memoryloss, long term and short term memory disorders, learning disorders,autism, depression, benign forgetfulness, children learning disorders,learning disorders, attention deficit disorder, neuronal reaction toviral infection, brain damage, hereditary myelin disorder of the CNS,epilepsy, perinatal asphyxia, asphyxia, anoxia, status epilepticus, andstroke, concussion (including post-concussion syndrome), baldness (suchas male pattern baldness), alopecia areata, addiction, clinicaldepression, neurofibromatosis, tuberous sclerosis, bipolar disorder,posttraumatic stress disorder, anxiety disorder, psychiatric disorderssuch as bi-polarism, schizophrenia and the like, narcolepsy/sleepdisorders (optionally including circadian rhythm disorders, insomnia,narcolepsy, or a combination of two or more of the foregoing); severanceof nerves or nerve damage, severance of the cerebrospinal nerve cord andany damage to brain or nerve cells, neurological deficits associatedwith AIDS, tics (for example Giles de la Tourette's syndrome),Huntington's chorea, schizophrenia, TBI, tinnitus, neuralgia, especiallytrigeminal neuralgia, neuropathic pain, inappropriate neuronal activityresulting in neurodysthesias in diseases such as diabetes, MS and motorneuron disease, ataxias, muscular rigidity (spasticity),temporomandibular joint dysfunction, atypical parkinsonian disorders,Down's syndrome, or a combination of two or more of the foregoingdisorders.

Certain embodiments may provide, for example, methods, tests, protocol,assays, kits, and/or systems for detecting, diagnosing, distinguishing,and/or excluding a neurological condition. In certain embodiments, forexample, the neurological condition may comprise MS. In certainembodiments, for example, the MS may comprise relapsing-remitting MS. Incertain embodiments, for example, the MS may comprise primaryprogressive MS. In certain embodiments, for example, the MS may compriseprogressive relapsing MS. In certain embodiments, for example, the MSmay comprise secondary progressive MS.

Certain embodiments may provide, for example, methods, tests, protocol,assays, kits, and/or systems for detecting, diagnosing, distinguishing,and/or excluding a neurological condition in a subject (for example asubject having symptoms indicative of the neurological condition or asubject having a history of the neurological condition or a relatedcondition). In certain embodiments, for example, the methods, tests,protocol, assays, kits, and/or systems may comprise calculating at leastone classification value based on a multivariate classification model ofthe concentrations of a plurality (for example 2, 3, 4, 5, 6, 7, 8, 9,10, or more than 10) of biomarkers (for example two or more (for examplethree or more) of NF-L, Tau, GFAP or UCH L1) in a liquid sample. Incertain embodiments, for example, the methods, tests, protocol, assays,kits, and/or systems may comprise assigning a risk of having (ordeveloping) neurological condition, comprising: comparing the at leastone classification value to at least one threshold value.

In certain embodiments, for example, the classification model may be astatistical model. In certain embodiments, for example, theclassification model may be a regression, linear regression, quadraticregression, polynomial regression, logistic regression, neural network,clustering model, principle component analysis, nearest neighborclassifier analysis, support vector machines, linear discriminantanalysis, quadratic discriminant analysis, decision trees, geneticalgorithm, classifier optimization using bagging, classifieroptimization using boosting, classifier optimization using the RandomSubspace Method, a projection pursuit, genetic programming, weightedvoting, or a combination of two or more of the foregoing.

In certain embodiments, for example, the classification model may be aregression model (for example a logistic regression model). In certainembodiments, for example, the regression model may include at least onecoefficient (for example 1 coefficient, 2 coefficients, or more than 2coefficients) for at least two (for example each or all) of thebiomarkers in a selected set of biomarkers (for example two or more (forexample three or more) of NF-L, Tau, GFAP or UCH L1). In certainembodiments, for example, the coefficients for the regression model maybe determined using a maximum likelihood algorithm. In certainembodiments, for example, the regression may be a logistic regression.In certain embodiments, for example, the logistic regression may be abinary logistic regression.

In certain embodiments, for example, the classification model may be aneural network. In certain embodiments, for example, the neural networkmay be constructed for a selected set of biomarkers (for example two ormore of NF-L, Tau, GFAP or UCH L1). In certain embodiments, for example,the neural network may be a two-stage regression. In certainembodiments, for example, the neural network may be a two stageclassification model. In certain embodiments, for example, the neuralnetwork may have a layered structure that includes a layer of inputunits connected by a layer of weights to a layer of output units. Incertain embodiments, for example, the neural network may be a multilayerneural network. In certain embodiments, for example, the multilayerneural network may comprise input layer, at least one hidden layer, andan output layer. In certain embodiments, for example, a single biaslayer may be connected to each layer other than the input layer.

In certain embodiments, for example, the classification model may becalibrated to determine the at least one threshold value at least basedon one or more data sets. In certain embodiments, for example, at leasta portion of the data set (for example at least 10%, at least 20%, atleast 30%, or at least 50% of the data set) may be derived frombiospecimens from healthy individuals (and/or individuals who do nothave a neurological condition nor a history of a neurologicalcondition). In certain embodiments, for example, at least a portion ofthe data set (for example at least 10%, at least 20%, at least 30%, orat least 50% of the data set) may be derived from biospecimens fromindividuals who have the neurological condition or a history of theneurological condition. In certain embodiments, for example, at least aportion of the data set (for example at least 10%, at least 20%, atleast 30%, or at least 50% of the data set) may comprise diagnosticresults from a CT scan and/or an MRI scan. In certain embodiments, forexample, the at least a portion of the data set (for example at least10%, at least 20%, at least 30%, or at least 50% of the data set) maycomprise positive diagnostic results for the neural condition (forexample CT-positive or MRI-positive results indicative of the neuralcondition). In certain embodiments, for example, at least a portion ofthe data set (for example at least 10%, at least 20%, at least 30%, orat least 50% of the data set) may comprise negative diagnostic resultsfor the neural condition (for example CT-negative or MRI-negativeresults indicative of the absence of the neural condition). In certainembodiments, for example, at least a portion of the data set (forexample at least 10%, at least 20%, at least 30%, or at least 50% of thedata set) may comprise mixed diagnostic results for the neural condition(for example CT-negative/MRI-positive or CT-positive/MRI-negativeresults).

In certain embodiments, for example, at least a portion of the data set(for example at least 10%, at least 20%, at least 30%, or at least 50%of the data set) may be obtained from individuals who may have beensubject to two or more diagnostic methods (for example a CT scan and anMRI scan). In certain embodiments, for example, at least a portion ofthe data set (for example at least 10%, at least 20%, at least 30%, orat least 50% of the data set) may be derived from biospecimens fromindividuals having positive results for the neurological condition in atleast one diagnostic method. In certain embodiments, for example, atleast a portion of the data set may (for example at least 10%, at least20%, at least 30%, or at least 50% of the data set) be derived frombiospecimens from individuals having a positive CT scan and a positiveMRI scan for the neurological condition. In certain embodiments, forexample, at least a portion of the data set (for example at least 10%,at least 20%, at least 30%, or at least 50% of the data set) may bederived from biospecimens from individuals having a positive CT scan anda negative MRI scan for the neurological condition. In certainembodiments, for example, at least a portion of the data set (forexample at least 10%, at least 20%, at least 30%, or at least 50% of thedata set) may be derived from biospecimens from individuals having anegative MRI scan and a positive MRI scan for the neurologicalcondition. In certain embodiments, for example, at least a portion ofthe data set (for example at least 10%, at least 20%, at least 30%, orat least 50% of the data set) may be derived from biospecimens fromindividuals having negative results for the neurological condition inthe two or more diagnostic methods. In certain embodiments, for example,at least a portion of the data set (for example at least 10%, at least20%, at least 30%, or at least 50% of the data set) may be derived frombiospecimens from individuals having a negative MRI scan and a negativeMRI scan for the neurological condition.

In certain embodiments, for example, the data set may be derived frombiospecimens from at least 25 healthy controls (for example at least 40healthy controls, at least 50 healthy controls, at least 75 healthycontrols, at least 100 healthy controls, or the data set may be derivedfrom biospecimens from at least 200 healthy controls. In certainembodiments, for example, the data set may be derived from biospecimensfrom at least 25 subjects exhibiting at least one indication from theneurological condition (for example at least 40 subjects, at least 50subjects, at least 75 subjects, at least 100 subjects, or the data setmay be derived from biospecimens from at least 200 subjects exhibitingat least one indication from the neurological condition.

In certain embodiments, for example, the neurological condition maycomprise TBI. In certain embodiments, for example, at least 10% (forexample at least 20%, at least 30%, or at least 50%) of the data set maycomprise data derived from biospecimens from subjects who have a GlasgowComa Score of 3-8, a Glasgow Coma Score of 9-12, and/or a Glasgow ComaScore of 13-15. In certain embodiments, for example, the data set may bederived from biospecimens (for example at least 50 biospecimens, atleast 75 biospecimens, at least 100 biospecimens, at least 125biospecimens, at least 150 biospecimens, or at least 200 biospecimens)obtained from the TRACK-TBI pilot study. In certain embodiments, forexample, the data set may be derived from biospecimens (for example atleast 50 biospecimens, at least 75 biospecimens, at least 100biospecimens, at least 125 biospecimens, at least 150 biospecimens, orat least 200 biospecimens) obtained from the Traumatic Head InjuryNeuroimaging Classification study (NCT01132937).

In certain embodiments, for example, at least a portion of the data setmay be derived from biospecimens from the subject. In certainembodiments, for example, at least a portion of the data set may bederived from biospecimens from the subject prior to developing (or priorto the detection of (for example by a healthcare provider)) symptomsindicative of the neurological condition, for example at least 5 minutesprior, at least 15 minutes prior to, at least 30 minutes prior to, atleast 45 minutes prior to, at least 1 hour prior to, at least 2 hoursprior to, at least 4 hours prior to, at least 6 hours prior to, at least12 hours prior to, at least 24 hours prior to, at least 48 hours priorto, at least 72 hours prior to, at least 96 hours prior to, at least 1week prior to, at least 2 weeks prior to, at least 3 weeks prior to, atleast 4 weeks prior to, at least 5 weeks prior to, at least 6 weeksprior to, at least 8 weeks prior to, or at least 12 weeks prior todeveloping (or prior to the detection of (for example by a healthcareprovider)) symptoms indicative of the neurological condition. In certainembodiments, for example, at least a portion of the data set may bederived from biospecimens from the subject subsequent to developing (orsubsequent to the detection of (for example by a healthcare provider))symptoms indicative of the neurological condition, for example at least5 minutes subsequent to, at least 15 minutes subsequent to, at least 30minutes subsequent to, at least 45 minutes subsequent to, at least 1hour subsequent to, at least 2 hours subsequent to, at least 4 hourssubsequent to, at least 6 hours subsequent to, at least 12 hourssubsequent to, at least 24 hours subsequent to, at least 48 hourssubsequent to, at least 72 hours subsequent to, at least 96 hourssubsequent to, at least 1 week subsequent to, at least 2 weekssubsequent to, at least 3 weeks subsequent to, at least 4 weekssubsequent to, at least 5 weeks subsequent to, at least 6 weekssubsequent to, at least 8 weeks subsequent to, or at least 12 weekssubsequent to developing (or subsequent to the detection of (for exampleby a healthcare provider)) symptoms indicative of the neurologicalcondition.

In certain embodiments, for example, the classification model may be atleast partially characterized by an ROC curve. In certain embodiments,for example, the ROC curve may provide one or more parameters toevaluate the sensitivity and specificity of the results of the methods,tests, protocol, assays, kits, and/or systems for detecting, diagnosing,distinguishing, and/or excluding a neurological condition. In certainembodiments, for example, the classification model may be calibrated todetermine the at least one threshold value at least based a preselectedtrue positive rate (i.e., sensitivity) and/or a preselected falsepositive rate (i.e., 1-specificity). In certain embodiments, forexample, the preselected true positive rate and/or preselected falsepositive rate may be selected from a point on the ROC curve. In certainembodiments, for example, the at least one threshold value may provide atrue positive rate (for example a true positive rate measured from theROC curve) of at least 60%, for example at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, orthe at least one threshold value may provide a true positive rate of100%. In certain embodiments, for example, the at least one thresholdvalue may provide a true positive rate of between 60% and 100%, forexample between 60% and 95%, between 70% and 95%, between 70% and 90%,or at least one threshold value may provide a true positive rate ofbetween 80% and 95%. In certain embodiments, for example, the at leastone threshold value may provide a false positive rate (for example afalse positive rate measured from the ROC curve) of less than 60%, forexample less than 50%, less than 40%, less than 30%, less than 20%, orthe at least one threshold value may provide a false positive rate ofless than 10%. In certain embodiments, for example, the at least onethreshold value may provide a false positive rate of between 10% and80%, for example between 10% and 50%, between 10% and 40%, between 10%and 30%, between 20% and 50%, or the at least one threshold value mayprovide a false positive rate of between 20% and 40%. In certainembodiments, for example, the at least one threshold value may provide atrue positive rate of at least 60% at a false positive rate of less than20%, for example a true positive rate of at least 65% at a falsepositive rate of less than 20%, a true positive rate of at least 70% ata false positive rate of less than 20%, a true positive rate of at least75% at a false positive rate of less than 20%, a true positive rate ofat least 80% at a false positive rate of less than 20%, a true positiverate of at least 85% at a false positive rate of less than 20%, or theat least one threshold value may provide a true positive rate of atleast a true positive rate of at least 90% at a false positive rate ofless than 20%. In certain embodiments, for example, the at least onethreshold value may provide a true positive rate of at least 60% at afalse positive rate of less than 30%, for example a true positive rateof at least 65% at a false positive rate of less than 30%, a truepositive rate of at least 70% at a false positive rate of less than 30%,a true positive rate of at least 75% at a false positive rate of lessthan 30%, a true positive rate of at least 80% at a false positive rateof less than 30%, a true positive rate of at least 85% at a falsepositive rate of less than 30%, or the at least one threshold value mayprovide a true positive rate of at least a true positive rate of atleast 90% at a false positive rate of less than 30%. In certainembodiments, for example, the at least one threshold value may provide atrue positive rate of at least 60% at a false positive rate of less than50%, for example a true positive rate of at least 65% at a falsepositive rate of less than 50%, a true positive rate of at least 70% ata false positive rate of less than 50%, a true positive rate of at least75% at a false positive rate of less than 50%, a true positive rate ofat least 80% at a false positive rate of less than 50%, a true positiverate of at least 85% at a false positive rate of less than 50%, or theat least one threshold value may provide a true positive rate of atleast a true positive rate of at least 90% at a false positive rate ofless than 50%.

In certain embodiments, for example, the ROC curve may have an AUC of atleast 0.95, for example at least 0.50, at least 0.55, at least 0.60, atleast 0.65, at least 0.70, at least 0.75, at least 0.80, at least 0.85,at least 0.875, at least 0.90, or the ROC curve may have an AUC of atleast 0.925. In certain embodiments, for example, the ROC curve may havean AUC of between 0.6 and 0.95, for example between 0.65 and 0.9,between 0.65 and 0.85, between 0.7 and 0.9, or the ROC curve may have anAUC of between 0.7 and 0.85.

Certain embodiments may provide, for example, methods, tests, assays,kits, or systems to quantify abnormally high (or abnormally depressed)levels of a plurality of biomarkers (for example a panel of biomarkers)indicative of a neurological condition in a subject. In certainembodiments, for example, the one or more biomarkers may be obtainedfrom physiological fluid (for example from a sample of venous orcapillary blood). In certain embodiments, for example, the physiologicalfluid may be limited to a single sample (for example a single sample ofblood obtained from the subject proximate the neurological condition).In certain embodiments, for example, the physiological fluid may be atleast 2 months old (for example between 2 months and 5 years old). Incertain embodiments, for example, the methods, tests, assays, kits, orsystems may comprise detecting at least one of the one or morebiomarkers at a molar concentration of less than 1 pmol/L. In certainembodiments, for example, concentrations of the plurality of biomarkersmay be determined in a series of assays performed on samples ofphysiological fluids taken from the subject at different times. Incertain embodiments, for example, results from the series of assays maybe used for diagnosis, prognosis, or monitoring to determine whether acondition resulting from the neurological condition may be improving orworsening.

Any of the methods, tests, assays, kits, or systems disclosed herein maycomprise preparing one or more calibration curves to convert an assayresult (for example signal readings or average exposed beadmeasurements) for one or more biomarkers into a measure of concentrationof the one or more biomarkers (for example a concentration expressed aspg/mL or pmole/L). In certain embodiments, for example, the one or morecalibration curves may comprise a plurality (for example a series) ofcalibration curves for a multiplex assay (for example a multiplex assayconfigured to quantify concentrations of a panel of biomarkers, such asa panel of biomarkers indicative of a neurological condition in asubject when present in a venous blood sample obtained from thesubject). In certain embodiments, for example, a series ofmulti-constituent calibrators having known concentrations of multiplebiomarkers may be prepared in a solution and assayed, and the resultingassay results correlated (for example by linear or nonlinear regression)to the known concentrations to obtain a calibration curve for eachbiomarker of the multiple biomarkers.

In certain embodiments, for example, the multi-constituent calibratorsmay comprise known (for example predetermined) concentrations of one ormore (for example all) of NF-L, GFAP, UCH L1, and Tau. In certainembodiments, for example, the multi-constituent calibrators may comprisepredetermined concentrations of one or more of purified NF-L, purifiedTau, purified GFAP antigen, and purified UCH L1. In certain embodiments,for example, the predetermined concentrations may be between 0.01% and20%, for example between 0.05% and 15%, or the predeterminedconcentrations may be at a concentration of between 0.1% and 10% (forexample purified NF-L at a concentration of 0.1-10%, purified Tau at aconcentration of 0.1-10%, purified GFAP at a concentration of 0.1-10%,and/or or purified UCH L1 at a concentration of 0.1-10%).

In certain embodiments, for example, the biomarker concentrations in theseries of multi-constituent calibrators may be selected based onexperimental design principles. In certain embodiments, for example, thebiomarker concentrations in the series of multi-constituent calibratorsmay be selected based on an experimental design comprising an orthogonalexperimental design. In certain embodiments, for example, the biomarkerconcentrations in the series of multi-constituent calibrators may beselected based on an experimental design comprising a principalcomponents analysis. In certain embodiments, for example, the biomarkerconcentrations in the series of multi-constituent calibrators may beselected based on an experimental design comprising a randomizedexperimental design. In certain embodiments, for example, the biomarkerconcentrations in the series of multi-constituent calibrators may beselected based on an experimental design comprising a factorialexperimental design. In certain embodiments, for example, the biomarkerconcentrations in the series of multi-constituent calibrators may beselected based on an experimental design comprising a response surfacemethodology. In certain embodiments, for example, the biomarkerconcentrations in the series of multi-constituent calibrators may beselected based on an experimental design comprising an optimalexperimental design. In certain embodiments, for example, the biomarkerconcentrations in the series of multi-constituent calibrators may beselected based on known cross-reactivities between one or more of thebiomarkers and one or more capture agents for the one or morebiomarkers.

In certain embodiments, for example, the series of multi-constituentcalibrators may comprise one or more (for example all) of compositionsA-H (concentrations expressed as pg/mL):

Composition NF-L GFAP UCH L1 Tau A 0 0 0 0 B 0.25-0.75 0.5-1.5 5-150.05-0.15 C 1.25-1.75 2-5 20-40  0.2-0.4 D 4-6  6-15 75-125 0.5-1.5 E10-20 20-40 250-350  2-5 F 25-75  75-125 800-1200  8-12 G 100-200250-350 2000-4000  20-40 H 400-500  800-1100 8000-10000  70-100

In certain embodiments, for example, the series of multi-constituentcalibrators may comprise one or more (for example all) of compositionsI-P (concentrations expressed as pg/mL):

Composition NF-L GFAP UCH L1 Tau I 0 0 0 0 J 0.25-0.75 0.5-1.5 5-150.05-0.15 K 1.25-1.75 2-5 20-40  0.2-0.4 L 4-6  6-15 75-125 0.5-1.5 M10-20 20-40 250-350  2-5 N 25-75  75-125 800-1200  8-12 O 100-200250-350 2000-4000  20-40 P 400-500  800-1100 8000-10000  70-100

In certain embodiments, for example, the series of multi-constituentcalibrators may comprise one or more (for example all) of compositionsQ-X (concentrations expressed as pg/mL):

Composition NF-L GFAP UCH L1 Tau Q 0 0 0 0 R 0.25-0.75 0.5-1.5 00.05-0.15 S 1.25-1.75 2-5 0 0.2-0.4 T 4-6  6-15 0 0.5-1.5 U 10-20 20-400 2-5 V 25-75  75-125 0  8-12 W 100-200 250-350 0 20-40 X 400-500 800-1100 0  70-100

In certain embodiments, for example, the series of multi-constituentcalibrators may comprise one or more (for example all) of compositionsY-AF (concentrations expressed as pg/mL):

Composition NF-L GFAP UCH L1 Tau Y 0 0 0 0 Z 0 0.5-1.5 0 0.05-0.15 AA 02-5 0 0.2-0.4 AB 0  6-15 0 0.5-1.5 AC 0 20-40 0 2-5 AD 0  75-125 0  8-12AE 0 250-350 0 20-40 AF 0  800-1100 0  70-100

In certain embodiments, for example, the series of multi-constituentcalibrators may comprise one or more (for example all) of compositionsAG-AN (concentrations expressed as pg/mL):

Composition NF-L GFAP UCH L1 Tau AG 0 0 0 0 AH 0.5 1 10 0.1 AI 1.5 3 300.3 AJ 5 10 100 1 AK 15 30 300 3 AL 50 100 1000 10 AM 150 300 3000 30 AN450 900 9000 90

In certain embodiments, for example, the series of multi-constituentcalibrators may comprise one or more (for example all) of compositionsAO-AV (concentrations expressed as pg/mL):

Composition NF-L GFAP UCH L1 Tau AO 0 0 0 0 AP 0.5 1 10 0.1 AQ 1.5 3 300.3 AR 5 10 100 1 AS 15 30 300 3 AT 50 100 1000 10 AU 150 300 3000 30 AV450 900 9000 90

In certain embodiments, for example, the series of multi-constituentcalibrators may comprise one or more (for example all) of compositionsAW-BD:

Composition NF-L GFAP UCH L1 Tau AW 0 0 0 0 AX 0.5 1 0 0.1 AY 1.5 3 00.3 AZ 5 10 0 1 BA 15 30 0 3 BB 50 100 0 10 BC 150 300 0 30 BD 450 900 090

In certain embodiments, for example, the series of multi-constituentcalibrators may comprise one or more (for example all) of compositionsBE-BL (concentrations expressed as pg/mL):

Composition NF-L GFAP UCH L1 Tau BE 0 0 0 0 BF 0 1 0 0.1 BG 0 3 0 0.3 BH0 10 0 1 BI 0 30 0 3 BJ 0 100 0 10 BK 0 300 0 30 BL 0 900 0 90

In certain embodiment, for example, at least one (for example all) ofthe multi-constituent calibrators may comprise one or more buffers (or abuffering system) (for example phosphate buffer), one or more ions (forexample Na⁺ and K⁺), one or more ionic salts (for example NaCl and KCl),one or more blocking agents, one or more surfactants, one or morecomplexing agents (for example ethylenediaminetetraacetic acid (EDTA) ora salt thereof), one or more anti-microbial agents (for example amixture of 2-methyl-3(2H)-isothiazolone and5-chloro-2-methyl-3(2H)-isothiazolone), or a combination of two or moreof the foregoing.

In certain embodiments, for example, the multi-constituent calibratorsmay comprise one or more buffers, or a buffering system (for example oneor more of the buffers or buffering systems disclosed herein or in oneof the INCORPORATED REFERENCES). In certain embodiments, for example,the one or more buffers may be selected from the group consisting ofcitrate buffers, phosphate buffers, borate buffers,tris(hydroxymethyl)aminomethane (Tris) buffers, barbital buffers,bicarbonates, as well as Good buffers (non-toxic to cells; not absorbedthrough cell membranes and feature pKa values at or near physiologicalpH) such as but not limited to N,N′-bis(2-hydroxyethyl)glycine (BIC IN),2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-1,3-propanediol(BISTRIS), 2-(cyclohexylamino)ethane-2-sulfonic acid (CHES),N-2-(hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES),N-(2-hydroxyethyl)piperazine-N′-3-propanesulfonic acid (HEPPS),morpholinoethanesulfonic acid (MES), morpholinopropanesulfonic acid(MOPS), piperazine-N, N′-bis(2-ethanesulfonic acid) (PIPES),N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES),N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS), andN-tris(hydroxymethyl)methylglycine (TRICINE), or a combination of two ormore of the foregoing. In certain embodiments, for example, themulti-constituent calibrators may comprise a buffering system comprisinga combination of buffers. In certain embodiments, for example, themulti-constituent calibrators may contain a phosphate buffer (forexample phosphate at a concentration of between 10 mM and 100 mM, suchas phosphate at a concentration of between 30 mM and 70 mM, or at aconcentration of 50 mM, or sodium phosphate (dibasic) at a concentrationof between 0.1% and 20%, such as sodium phosphate (dibasic) at aconcentration of between 0.5% and 15%, or at a concentration of between1% and 10%, and/or potassium phosphate (monobasic) at a concentration ofbetween 0.01% and 10%, such as potassium phosphate (monobasic) at aconcentration of between 0.5% and 5% or at a concentration of between0.1% and 1%).

In certain embodiments, for example, the one or more ions may compriseone or more ions (for example one or more of the ions disclosed hereinor in one of the INCORPORATED REFERENCES). In certain embodiments, forexample, the one or more ions may be selected from the group consistingof Li⁺, Na⁺, K⁺, Mg²⁺, Ca²⁺, Cu²⁺, Mn²⁺, Fe²⁺, Fe³⁺, Cl⁻, Br⁻,carbonate, hydrogen carbonate, hydrogen sulfate, hydrogen sulfite,sulfate, sulfite, monohydrogen phosphate, dihydrogen phosphate, nitrate,nitrite, permanganate, silicate, sulphates, pyrosulphates,pyrophosphates, citrates, cacodylates, or a combination of two or moreof the foregoing. In certain embodiments, for example, the one or moreions may comprise Cl⁻ (for example Cl⁻ at a concentration of between 100mM and 200 mM, such as Cl⁻ at a concentration of between 120 mM and 160mM, or at a concentration of 141.7 mM). In certain embodiments, forexample, the one or more ions may comprise Na⁺ (for example Na⁺ at aconcentration of between 100 mM and 200 mM, such as Na⁺ at aconcentration of between 120 mM and 160 mM, or at a concentration of 137mM). In certain embodiments, for example, the one or more ions maycomprise K⁺ (for example K⁺ at a concentration of between 1 mM and 5 mM,such as K⁺ at a concentration of between 2 mM and 3 mM, or at aconcentration of 2.7 mM). In certain embodiments, for example, the oneor more ions may comprise Mg²⁺ (for example Mg²⁺ at a concentration ofbetween 0.1 mM and 5 mM, such as Mg²⁺ at a concentration of between 0.5mM and 2.5 mM, or at a concentration of 1 mM). In certain embodiments,for example, the one or more ions may comprise phosphate (for examplephosphate at a concentration of between 10 mM and 100 mM, such asphosphate at a concentration of between 30 mM and 70 mM, or at aconcentration of 50 mM).

In certain embodiments, for example, the ions may form one or more ionicsalts (for example one or more of the ionic salts disclosed herein or inone of the INCORPORATED REFERENCES). In certain embodiments, forexample, the one or more ionic salts may be selected from the groupconsisting of KCl, NaCl, MgCl₂, KH₂PO₄, K₂HPO₄, NaH₂PO₄, Na₂HPO₄, NaHCO₃and other suitable ionic salts. In certain embodiments, for example, theone or more ionic salts may comprise KCl (for example KCl at aconcentration of between 1 mM and 5 mM, such as KCl at a concentrationof between 2 mM and 3 mM, or at a concentration of 2.7 mM or KCl at aconcentration of between 0.001% and 1%, such as KCl at a concentrationof between 0.005% and 0.5%, or at a concentration of between 0.01% and0.1%). In certain embodiments, for example, the one or more ionic saltsmay comprise NaCl (for example NaCl at a concentration of between 100 mMand 200 mM, such as NaCl at a concentration of between 120 mM and 160mM, or at a concentration of 137 mM, or NaCl at a concentration ofbetween 0.05% and 20%, such as NaCl at a concentration of between 0.1%and 10%, or at a concentration of 0.5% and 5%). In certain embodiments,for example, the one or more ionic salts may comprise MgCl₂ (for exampleMgCl₂ at a concentration of between 0.1 mM and 5 mM, such as MgCl₂ at aconcentration of between 0.5 mM and 2.5 mM, or at a concentration of 1mM).

In certain embodiments, for example, the multi-constituent calibratorsmay comprise one or more surfactants (for example one or more of thesurfactants disclosed herein or in one of the INCORPORATED REFERENCES).In certain embodiments, for example, the one or more surfactants maycomprise one or more ionic surfactants. In certain embodiments, forexample, the one or more surfactants may comprise one or more nonionicsurfactants. In certain embodiments, for example, the one or moresurfactants may comprise a glycidyl surfactant (for example 10Gsurfactant (for example 10G surfactant at a concentration of between0.01% and 1%, or at a concentration of 0.1%)). In certain embodiments,for example, the one or more surfactants may comprise one or moredetergents (for example one or more of the detergents disclosed hereinor in one of the INCORPORATED REFERENCES). In certain embodiments, forexample, the one or more detergents may be selected from the groupconsisting of nonionic, cationic, anionic and amphoteric forms. Incertain embodiments, for example, the one or more detergents may beselected from the group consisting of polyoxyethylene sorbitan alcoholdetergents (i.e., the Tween series), polyoxyethylene alcohols such asthe non-ionic, non-denaturing detergent sold under the trademarkNonidet™ P-40 or polyoxyethylene ethers such as Triton™ X-100, or acombination of two or more of the foregoing. In certain embodiments, forexample, the one or more detergents may comprise Triton™ X-100 (forexample Triton™ X-100 at a concentration of between 0.005% and 2% (forexample at a concentration of 0.5%), between 0.1% and 1%, or themulti-constituent calibrators may comprise Triton™ X-100 at aconcentration of between 0.25% and 0.75%). In certain embodiments, forexample, the surfactant may be at a concentration of between 0.005% and2%, between 0.01% and 1.5%, or at a concentration of between 0.1% and1%.

In certain embodiments, multi-constituent calibrators may comprise oneor more immunoglobulins (or antibodies or fragments thereof) (forexample one of the immunoglobulins (or antibodies or fragments thereof)thereof disclosed herein or in one of the INCORPORATED REFERENCES). Incertain embodiments, for example, the one or more immunoglobulins may beselected from the classes consisting of IgA, IgD, IgE, IgG, IgM, asub-class of one or more of the foregoing, or a combination of two ofmore of the foregoing. In other embodiments, for example, the one ormore immunoglobulins may not belong to any particular class. In certainembodiments, for example, the one or more immunoglobulins may containdifferent heavy-chain constant domains that correspond to the differentclasses of immunoglobulins, such as alpha, delta, epsilon, gamma, andmu, respectively. In other embodiments, for example, the one or moreimmunoglobulins may contain heavy-chain constant domains that do notcorrespond to any particular class of immunoglobulins. In certainembodiments, for example, the one or more immunoglobulins may comprisebut may be not limited to an immunoglobulin of any subclasses (isotypes)in the major classes, such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. Incertain embodiments, for example, the one or more immunoglobulins may benot of any subclasses (isotypes) in the major classes. In certainembodiments, for example, the one or more immunoglobulins may be ofmurine, rat, human, bovine, goat, rabbit, or sheep origin. In certainembodiments, for example, the one or more immunoglobulins may comprise anatural immunoglobulin. In certain embodiments, for example, the one ormore immunoglobulins may comprise a genetically modified immunoglobulin.In certain embodiments, for example, the genetically modifiedimmunoglobulin may be a chimeric or humanized immunoglobulin. In certainembodiments, for example, the one or more immunoglobulins may beselected from the group consisting of human IgA, human IgD, human IgE,human IgG, human IgM, murine IgA, murine IgD, murine IgE, murine IgG,murine IgM, rat IgA, rat IgD, rat IgE, rat IgG, rat IgM, bovine IgA,bovine IgD, bovine IgE, bovine IgG, bovine IgM, or a combination of twoor more of the foregoing. In certain embodiments, for example, the oneor more immunoglobulins may comprise human IgG (for example human IgG ata concentration of between 1 mg/mL and 10 mg/mL, of between 3 mg/mL and7 mg/mL, or at a concentration of 5 mg/mL).

In certain embodiments, for example, the multi-constituent calibratormay comprise one or more blocking agents (for example one or more of theblocking agents disclosed herein or in one of the INCORPORATEDREFERENCES). In certain embodiments, for example, the one or moreblocking agents may be configured to prevent non-specific binding to oneor more assay surfaces, such as a microtiter well surface. In certainembodiments, for example, the one or more blocking agents may beconfigured to prevent non-specific binding to one or more targetanalytes (for example NF-L, GFAP, etc.). In certain embodiments, forexample, the one or more blocking agents may be configured to preventnon-specific binding to a capture agent or a detection agent (forexample the capture agent or detection agent described herein). Incertain embodiments, for example, the one or more blocking agents may beselected from the group consisting of detergents (for example Triton™X-100 and a Tween), BSA, ovalbumin, glucose, other sugars, polyethyleneglycol, dextran, lysozyme, and poly L-lysine, or a combination of two ormore of the foregoing. In certain embodiments, for example, the one ormore blocking agents may comprise BSA (for example BSA at aconcentration of between 0.005% and 0.05% (for example 0.02%), between0.05% and 0.5%, between 0.5% and 1%, between 1% and 5%, or at aconcentration of 2%).

In certain embodiments, for example, one of the one or moreimmunoglobulins (or antibodies) may be a heterophilic interferenceinhibitor. In certain embodiments, for example, human IgG may be aheterophilic interference inhibitor. In certain embodiments, forexample, one of the one or more immunoglobulins (or antibodies) may be aheterophilic interference molecule. In certain embodiments, for example,human IgG may be a heterophilic interference molecule. In certainembodiments, for example, the multi-constituent calibrators may compriseone or more interference molecules (for example one or more of theinterference molecules disclosed herein or in one of the INCORPORATEDREFERENCES). In certain embodiments, for example, the one or moreinterference molecules may be selected from the group consisting ofHAGA, HAMA, HARA, HASA, rheumatoid factor, or a combination of two ormore of the foregoing.

In certain embodiments, for example, the one or more blocking agents maycomprise a component that is a heterophilic interference inhibitor whenadded to a predetermined type of sample (for example a blood sample,urine sample, CSF sample, etc.). In certain embodiments, for example,the heterophilic interference inhibitor may block one or moreheterophilic interference molecules from binding to one or more of theanalytes disclosed herein, such as NF-L, GFAP, UCH L1 and/or Tau. Incertain embodiments, for example, the heterophilic interferenceinhibitor may block one or more heterophilic interference molecules frombinding a capture agent or a detection agent (for example the captureagents or detection agents described herein). In certain embodiments,for example, at least one of the one or more inhibitors may be selectedfrom the group consisting of BSA, protein L, collagen, PEG4000/6000,whole normal animal serum (for example mouse serum, rat serum, goatserum, rabbit serum, sheep serum), an animal based IgG aggregate (forexample mouse IgG, rat IgG, rabbit IgG, goat IgG, sheep IgG), and anantibody derived from goat, mouse, rabbit or sheep that recognizes aHAGA, HAMA, HARA, HASA, rheumatoid factor, Superchemiblock™ heterophileblocking agent (Millipore, Billerica, Mass.), TRU Block™ (MeridianBioscience, Memphis, Tenn.), immunoglobulin-inhibiting reagent (IIR;Bioreclamation, Inc., Westbury, N.Y.), heterophile blocking tubes(Scantibodies Laboratory, Santee, Calif.), StabliGuard immunoassaystabilizer (SurModics, Inc., Eden Prairie, Minn.), one of the blockingagents disclosed in the INCORPORATED REFERENCES, or a combination of twoor more of the foregoing. In certain embodiments, for example, theblocking agent may be an interference blocker at a concentration ofbetween 0.001% and 1%, between 0.005% and 0.5%, or at a concentration ofbetween 0.01% and 0.1%.

In certain embodiments, for example, the heterophilic interferenceinhibitor may comprise an antibody (for example IgG, IgG, IgM, IgE orIgD), for example of animal (for example mouse, rabbit, sheep, goat,donkey, and other suitable animals) origin. In certain embodiments, forexample, the antibody may specifically bind and neutralize one or moreheterophilic antibodies, one or more rheumatoid factors, or one or moreother interference molecules. For example, the attachment of theimmunoglobulin to a heterophilic antibody prevents the heterophilicantibody from binding (capturing) an antibody that may be specific foran analyte or a detection antibody. The one or more heterophilicinterference inhibitors may be one or more antibodies that may not bindto one or more analytes or one or more affinity antibodies that may bespecific for (for example may specifically bind to) the one or moreanalytes.

In certain embodiments, for example, the one or more binding agents maycomprise one or more heterophile antibody blocking agents (for exampleone or more of the heterophile antibody blocking agents disclosed hereinor in one of the INCORPORATED REFERENCES). In certain embodiments, forexample, the one or more binding agents may comprise Superchemiblock™(for example Superchemiblock™ at a concentration of between 10 pg/mL and100 pg/mL, such as Superchemiblock™ at a concentration of between 30pg/mL and 70 pg/mL, at a concentration of 50 pg/mL, between 0.005% and0.05% (for example 0.02%), between 0.05% and 0.5%, between 0.5% and 1%,between 1% and 5%, or at a concentration of 0.05%).

In certain embodiments, for example, the one or more binding agents maycomprise one or more human anti-mouse antibody (HAMA) blockers (forexample one of the HAMA blockers disclosed herein or in one of theINCORPORATED REFERENCES). In certain embodiments, for example, the oneor more HAMA blockers may comprise TRU Block™ (for example TRU Block™ ata concentration of between 1 mcg/mL and 100 mcg/mL, such as TRU Block™at a concentration of between 5 mcg/mL and 15 mcg/mL, or at aconcentration of 10 mcg/mL).

In certain embodiments, for example, the one or more binding agents maybind to one or more interference molecules. In certain embodiments, forexample, more than one type of binding agent may bind to the sameinterference molecule. In certain embodiments, for example, the one ormore blocking agents may comprise a first binding agent and a secondbinding agent. In certain embodiments, for example, the first bindingagent and the second binding agent may bind to the same interferencemolecule. In certain embodiments, for example, the first binding agentand the second binding agent may not bind to the same interferencemolecule. In certain embodiments, for example, the one or more blockingagents may comprise a plurality (for example 2, 3, 4, 5, 6, 7, 8, 9, 10,or more than 10) of different types of binding agents. In someembodiments, the one or more binding agents may bind to a plurality (forexample 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10) of different typesof interference molecules.

In certain embodiments, for example, the more than one binding agentsmay be utilized in a one-to-one ratio. For instance, the first bindingagent and the second binding agent may present at equal or approximatelyequal concentrations (for example molar concentrations). In certainembodiments, for example, the more than one binding agents may bepresent at different concentrations (for example molar concentrations).In certain embodiments, for example, the first binding agent may bepresent at a concentration that is at least 1 times greater than theconcentration of the second binding agent, for example at least 2 times,at least 3 times, at least 4 times, at least 5 times, or the firstbinding agent may be present at a concentration that is at least 6 timesgreater than the concentration of the second binding agent. In certainembodiments, for example, the one or more binding agents may be aplurality of binding agents comprising TRU Block™ and Superchemiblock™(for example TRU Block™ and Superchemiblock™ at a ratio of between 1:1and 1:10, such as TRU Block™ and Superchemiblock™ at a ratio of between1:3 and 1:7, or at a ratio of 1:5).

In certain embodiments, for example, the multi-constituent calibratorsmay comprise one or more sugars (for example one or more of the sugarsdisclosed herein or in one of the INCORPORATED REFERENCES). In certainembodiments, for example, the one or more sugars may comprise dextrose(for example dextrose at a concentration of between 0.005% and 0.05%(for example at a concentration of 0.02%), between 0.05% and 0.5%,between 0.5% and 1%, between 1% and 5%, or at a concentration of 0.06%).

In certain embodiments, for example, the multi-constituent calibratorsmay comprise BgG (for example BgG at a concentration of between 0.005%and 0.05% (for example at a concentration of 0.02%), between 0.05% and0.5%, between 0.5% and 1%, between 1% and 5%, or at a concentration of0.01%).

In certain embodiments, for example, the multi-constituent calibratorsmay comprise urea (for example urea at a concentration of between 0.5 mMand 100 mM (for example at a concentration of 10 mM), between 1 mM and20 mM, between 1 mM and 10 mM, or at a concentration of between 3 mM and7 mM, or urea at a concentration of between 0.001% and 1% (for example aconcentration of 0.05%), between 0.005% and 0.5%, or at a concentrationof 0.01%).

In certain embodiments, for example, the multi-constituent calibratorsmay comprise one or more complexing agents that are capable of forming acomplex with a metal ion (for example one of the complexing agentsdisclosed herein or in one of the INCORPORATED REFERENCES). In certainembodiments, for example, the one or more complexing agents may beselected from the group consisting of EDTA, deferoxamine (DESFERAL),NTA, β-alaninediacetic acid (β-ADA), diethylenetriaminepentaacetic acid(DTPA), diethylenetriaminepentakis-methylenephosphonic acid (DTPMP),nitrilotriacetic acid (NTA), N-bis[2-1,2-dicarboxyethoxy)ethyl]glycine(BCAS), N-bis[2-1,2-dicarboxyethoxy)ethyl]aspartic acid (BCA6),N-(hydroxyethyl)-ethylenediaminetriacetic acid (HEDTA), gluconic acidand tetracis(2-hydroxypropyl)ethylenediamine (THPED), other suitablecomplexing agents, or a combination of two or more of the foregoing. Incertain embodiments, for example, the complexing agent may comprise EDTAor a salt of EDTA (for example EDTA or salt of EDTA at a concentrationof between 1 mM and 50 mM, of between 1 mM and 10 mM, or at aconcentration of 5 mM, or EDTA or salt of EDTA at a concentration of0.02-10%, 0.1-5%, or at a concentration of 0.2-2%). In certainembodiments, for example, the salt of EDTA may be sodium edetate or EDTAdisodium salt dihydrate.

In certain embodiments, for example, the multi-constituent calibratorsmay comprise one or more anti-microbial agents (for example one of theanti-microbial agents disclosed herein or in one of the INCORPORATEDREFERENCES). In certain embodiments, for example, the one or moreanti-microbial agents may be selected from the group consisting ofbenzalkonium chloride, sodium azide, sodium fluoride, phenoxyethanol,sodium dehydroacetate, chlorobutanol, phenylethanol, 4-chloroxylenol,1-hydroxypyridine-2-thione, paraben derivatives, glutaraldehyde,formaldehyde, nalidixic acid, sodium-2-pyridinethiol-1-oxide (sold underthe trademark Sodium Omadine™), the bactericidal antimicrobial soldunder the trademark Triadine™ 3, the bactericidal antimicrobial soldunder the trademark Triadine™ 10, combinations of5-chloro-2-methyl-4-isothiazolin-2-one, 2-methyl-4-isothiazolin-3-one,5-bromo-5-nitro-1,3-dioxane, derivatives of each of these compounds, ora combination of two or more of the foregoing. In certain embodiments,for example, the one or more anti-microbial agents may be commerciallyavailable and selected from the group consisting of reagents comprisingaqueous combinations of 5-chloro-2-methyl-4-methyl-4-isothiazolin-2-oneand 2-methyl-4-isothiazolin-3-one (Supelco, under the trademarkProclin), for example, the preservative sold under the trademarkProClin™ 150 reagent (Supelco, an aqueous mixture of 1.15% of5-chloro-2-methyl-4-isothiazolin-3-one and 0.35% of2-methyl-4-isothiazolin-3-one), the preservative sold under thetrademark ProClin™ 300 reagent (Supelco, a mixture of 2.3% of5-chloro-2-methyl-4-isothiazolin-3-one and 0.7% of2-methyl-4-isothiazolin-3-one in a solvent consisting of a modifiedglycol and alkyl carboxylate), the preservative sold under the trademarkProclin™ 5000 reagent (Supelco, 2-methyl-4-isothiazolin-3-one in adipropylene glycol solvent), the preservative sold under the trademarkBronidox® L reagent (Cognis Corporation, biocide5-bromo-5-nitro-1,3-dioxane), or a combination of two or more of theforegoing. In certain embodiments, for example, the multi-constituentcalibrators may comprise a combination of two or more anti-microbialagents in any ratio effective to combat microbial growth. In certainembodiments, for example, the one or more anti-microbial agents maycomprise benzalkonium chloride. In certain embodiments, for example, theone or more anti-microbial agents may comprise ProClin™ 300 (for exampleProClin™ 300 at a concentration of between 0.005% and 0.05% (for exampleat a concentration of 0.02%), between 0.05% and 0.5%, between 0.5% and1%, between 1% and 5%, or at a concentration of 2%). In certainembodiments, for example, the one or more anti-microbial agents maycomprise a mixture of 2-methyl-3(2H)-isothiazolone and5-chloro-2-methyl-3(2H)-isothiazolone (for example the mixture having atotal concentration of between 0.01% and 5%, between 0.05% and 1%, orhaving a total concentration of between 0.1% and 0.5%).

In certain embodiments, for example, the multi-constituent calibratorsmay have a pH of between 2.5 and 10 (for example a pH of between 5 and9, of between 7 and 8, a pH of 7.4, or an approximately neutral pH).

In certain embodiments, for example, the multi-constituent calibratorsmay comprise phosphate buffered saline, KCl, BSA, 10G Surfactant, TRUBlock™, EDTA, and an anti-microbial agent. In certain embodiments, forexample, the multi-constituent calibrators may comprise: phosphatebuffered saline, KCl at a concentration of between 3 mM and 5 mM, BSA ata concentration of between 1% and 3%, 10G surfactant at a concentrationof between 0.05% and 0.15%, TRU Block™ at a concentration of between 5mcg/mL and 15 mcg/mL, EDTA or salt of EDTA at a concentration of between3 mM and 7 mM, and an anti-microbial agent, wherein themulti-constituent calibrators may have a pH of between 7 and 8. Incertain embodiments, for example, the multi-constituent calibrators maycomprise: phosphate buffered saline, KCl at a concentration of 2.7 mM,BSA at a concentration of 2%, 10G surfactant at a concentration of 0.1%,TRU Block™ at a concentration of 10 mcg/mL, EDTA or salt of EDTA at aconcentration of 5 mM, and an anti-microbial agent, wherein themulti-constituent calibrators may have a pH of 7.4. In certainembodiments, for example, the multi-constituent calibrators maycomprise: phosphate buffered saline, dextrose at a concentration of0.06%, BSA at a concentration of 0.02%, BgG at a concentration of 0.01%,urea at a concentration of 5 mM, Triton™ X-100 at a concentration of0.5%, TRU Block™ at a concentration of 10 mcg/mL, Superchemiblock™ at aconcentration of 0.05%, and an anti-microbial agent, wherein themulti-constituent calibrators may have a pH of 7.4.

In certain embodiments, for example, the multi-constituent calibratorsmay comprise water, sodium phosphate (dibasic), potassium phosphate(monobasic), NaCl, KCl, BSA, a surfactant (for example 10G Surfactant),an interference blocker (for example TRU Block™), EDTA disodium saltdihydrate, and an anti-microbial agent (or a mixture of anti-microbialagents). In certain embodiments, for example, the multi-constituentcalibrators may comprise: water at a concentration between 90% and 100%,sodium phosphate (dibasic) at a concentration between 1% to 10%,potassium phosphate (monobasic) at a concentration between 0.1% and 1%,NaCl at a concentration between 0.5% and 5%, KCl at a concentration ofbetween 0.01% and 0.1%, BSA at a concentration of between 0.01% and0.1%, a surfactant at a concentration of between 0.1% and 1%, aninterference blocker at a concentration of between 0.01% and 0.1%, EDTAor salt of EDTA at a concentration of between 0.2% and 2%, and ananti-microbial agent at a concentration between 0.1% and 0.5%, whereinthe multi-constituent calibrators may have a pH of between 7 and 8.

In certain embodiments, for example, the multi-constituent calibratorsmay comprise: phosphate, one or more salts (for example NaCl and/orKCl), BSA, 10G Surfactant, and EDTA. In certain embodiments, forexample, the multi-constituent calibrators may comprise: 20-200 mMphosphate, 50-250 mM NaCl, 1-5 mM KCl, 0.5-5% BSA, 0.05-0.25% 10GSurfactant, 1-10 mcg/mL TRU block, 0.005-0.25% ProClin™ 300, and 0.5-20mM EDTA, wherein the multi-constituent calibrators may have a pH of6-8.5. In certain embodiments, for example, the multi-constituentcalibrators may comprise human IgG (for example human IgG at aconcentration of 0.5-20 mg/mL (for example a concentration of 5 mg/mL).In certain embodiments, for example, the multi-constituent calibratorsmay comprise: 50 mM phosphate, 137 mM NaCl, 2.7 mM KCl, 2% BSA, 0.1% 10GSurfactant, 10 mcg/mL TRU Block™, 0.05% ProClin™ 300, and 5 mM EDTA,wherein the multi-constituent calibrators may have a pH of 7.4.

Any of the methods, tests, assays, kits, or systems disclosed herein maycomprise a sample diluent configured to dilute the liquid sample into aworking range (for example a working range of analyte concentrations orworking range of fluid properties of such as viscosity of the dilutedliquid sample) for performing an immunoassay. In certain embodiments,for example, the sample diluent may be mixed with the liquid sample tomake a diluted liquid sample (for example a 2× dilution, 4× dilution, 8×dilution, 16× dilution, 32× dilution, 64× dilution, or 128× dilution)suitable for analysis. In certain embodiments, for example, the diluentmay be an aqueous diluent.

In certain embodiments, for example, the diluent may comprise aplurality of components selected from the group consisting of one ormore buffers (or a buffering system), one or more ions, one or moreionic salts, one or more blocking agents, one or more surfactants, oneor more complexing agents, and one or more anti-microbial agents. Thediluent may contain any of the buffers (or buffer systems), ions, ionicsalts, blocking agents, surfactants, complexing agents, immunoglobulins,and/or anti-microbial agents disclosed herein (for example disclosed inthe discussion of the multi-component calibrators) or in one of theINCORPORATED REFERENCES.

In certain embodiments, for example, the diluent may comprise one ormore immunoglobulins (or antibodies or fragments thereof) (for exampleone of the immunoglobulins (or antibodies or fragments thereof) thereofdisclosed herein or in one of the INCORPORATED REFERENCES). In certainembodiments, for example, the one or more immunoglobulins may beselected from the classes consisting of IgA, IgD, IgE, IgG, IgM, asub-class of one or more of the foregoing, or a combination of two ofmore of the foregoing. In other embodiments, for example, the one ormore immunoglobulins may not belong to any particular class. In certainembodiments, for example, the one or more immunoglobulins may containdifferent heavy-chain constant domains that correspond to the differentclasses of immunoglobulins, such as alpha, delta, epsilon, gamma, andmu, respectively. In other embodiments, for example, the one or moreimmunoglobulins may contain heavy-chain constant domains that do notcorrespond to any particular class of immunoglobulins. In certainembodiments, for example, the one or more immunoglobulins may comprisebut may be not limited to an immunoglobulin of any subclasses (isotypes)in the major classes, such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. Incertain embodiments, for example, the one or more immunoglobulins may benot of any subclasses (isotypes) in the major classes. In certainembodiments, for example, the one or more immunoglobulins may be ofmurine, rat, human, bovine, goat, rabbit, or sheep origin. In certainembodiments, for example, the one or more immunoglobulins may comprise anatural immunoglobulin. In certain embodiments, for example, the one ormore immunoglobulins may comprise a genetically modified immunoglobulin.In certain embodiments, for example, the genetically modifiedimmunoglobulin may be a chimeric or humanized immunoglobulin. In certainembodiments, for example, the one or more immunoglobulins may beselected from the group consisting of human IgA, human IgD, human IgE,human IgG, human IgM, murine IgA, murine IgD, murine IgE, murine IgG,murine IgM, rat IgA, rat IgD, rat IgE, rat IgG, rat IgM, bovine IgA,bovine IgD, bovine IgE, bovine IgG, bovine IgM, or a combination of twoor more of the foregoing. In certain embodiments, for example, the oneor more immunoglobulins may comprise human IgG (for example human IgG ata concentration of between 1 mg/mL and 10 mg/mL, of between 3 mg/mL and7 mg/mL, or at a concentration of 5 mg/mL).

In certain embodiments, for example, the diluent may comprise phosphatebuffered saline, KCl, BSA, 10G Surfactant, TRU Block™, EDTA, and ananti-microbial agent. In certain embodiments, for example, the diluentmay comprise: phosphate buffered saline, KCl at a concentration ofbetween 3 mM and 5 mM, BSA at a concentration of between 1% and 3%, 10Gsurfactant at a concentration of between 0.05% and 0.15%, TRU Block™ ata concentration of between 5 mcg/mL and 15 mcg/mL, EDTA or salt of EDTAat a concentration of between 3 mM and 7 mM, and an anti-microbialagent, wherein the diluent may have a pH of between 7 and 8. In certainembodiments, for example, the diluent may comprise: phosphate bufferedsaline, KCl at a concentration of 2.7 mM, BSA at a concentration of 2%,10G surfactant at a concentration of 0.1%, TRU Block™ at a concentrationof 10 mcg/mL, EDTA or salt of EDTA at a concentration of 5 mM, and ananti-microbial agent, wherein the diluent may have a pH of 7.4. Incertain embodiments, for example, the diluent may comprise: phosphatebuffered saline, dextrose at a concentration of 0.06%, BSA at aconcentration of 0.02%, BgG at a concentration of 0.01%, urea at aconcentration of 5 mM, Triton™ X-100 at a concentration of 0.5%, TRUBlock™ at a concentration of 10 mcg/mL, Superchemiblock™ at aconcentration of 0.05%, and an anti-microbial agent, wherein the diluentmay have a pH of 7.4.

In certain embodiments, for example, the sample diluent may comprise:phosphate, one or more ionic salts (for example NaCl, KCl, and/orMgCl₂), BSA, a sugar (for example dextrose), BgG, urea, Triton™ X-100,TRU Block™, Superchemiblock™ and ProClin™ 300. In certain embodiments,for example, the sample diluent may comprise an IgG (for example a humanIgG). In certain embodiments, for example, the sample diluent maycomprise: 20-200 mM phosphate, 50-250 mM NaCl, 1-5 mM KCl, 0.005-0.1%BSA, 0.25-5 mM MgCl₂, 0.005-0.25% dextrose, 0.001-0.05% BgG, 0.5-10 mMurea, 0.05-2% Triton™ X-100, 1-100 mcg/mL TRU Block™, 1-100 mcg/mLSuperchemiblock™, 0.005-0.25% ProClin™ 300, and 0.5-20 mg/mL human IgG,wherein the sample diluent may have a pH of 6-8.5. In certainembodiments, for example, the sample diluent may comprise: 50 mMphosphate, 137 mM NaCl, 2.7 mM KCl, 0.02% BSA, 1 mM MgCl₂, 0.06%dextrose, 0.01% BgG, 5 mM urea, 0.5% Triton™ X-100, 10 mcg/mL TRUBlock™, 50 mcg/mL Superchemiblock™, and 0.05% ProClin™ 300, wherein thesample diluent may have a pH of 7.4. In certain embodiments, forexample, the sample diluent may comprise: 50 mM phosphate, 137 mM NaCl,2.7 mM KCl, 0.02% BSA, 1 mM MgCl₂, 0.06% dextrose, 0.01% BgG, 5 mM urea,0.5% Triton™ X-100, 10 mcg/mL TRU Block™, 50 mcg/mL Superchemiblock™,0.05% ProClin™ 300, and 5 mg/mL human IgG, wherein the sample diluentmay have a pH of 7.4.

In certain embodiments, for example, (a) the multi-constituentcalibrators may comprise: phosphate, one or more salts (for example NaCland/or KCl), BSA, 10G Surfactant, and EDTA; and (b) the sample diluentmay comprise: phosphate, one or more ionic salts (for example NaCl, KCl,and/or MgCl₂), BSA, a sugar (for example dextrose), BgG, urea, Triton™X-100, TRU Block™, Superchemiblock™, ProClin™ 300, and optionally humanIgG. In certain embodiments, for example, (a) the multi-constituentcalibrators may comprise: 20-200 mM phosphate, 50-250 mM NaCl, 1-5 mMKCl, 0.5-5% BSA, 0.05-0.25% 10G Surfactant, 1-10 mcg/mL TRU block,0.005-0.25% ProClin™ 300, and 0.5-20 mM EDTA, wherein themulti-constituent calibrators may have a pH of 6-8.5; and (b) the samplediluent may comprise: 20-200 mM phosphate, 50-250 mM NaCl, 1-5 mM KCl,0.005-0.1% BSA, 0.25-5 mM MgCl₂, 0.005-0.25% dextrose, 0.001-0.05% BgG,0.5-10 mM urea, 0.05-2% Triton™ X-100, 1-100 mcg/mL TRU Block™, 1-100mcg/mL Superchemiblock™, 0.005-0.25% ProClin™ 300, and optionally 0.5-20mg/mL human IgG, wherein the sample diluent may have a pH of 6-8.5. Incertain embodiments, for example, (a) the multi-constituent calibratorsmay comprise: 50 mM phosphate, 137 mM NaCl, 2.7 mM KCl, 2% BSA, 0.1% 10GSurfactant, 10 mcg/mL TRU Block™, 0.05% ProClin™ 300, and 5 mM EDTA,wherein the multi-constituent calibrators may have a pH of 7.4; and (b)the sample diluent may comprise: 50 mM phosphate, 137 mM NaCl, 2.7 mMKCl, 0.02% BSA, 1 mM MgCl₂, 0.06% dextrose, 0.01% BgG, 5 mM urea, 0.5%Triton™ X-100, 10 mcg/mL TRU Block™, 50 mcg/mL Superchemiblock™, 0.05%ProClin™ 300, and optionally 5 mg/mL human IgG, wherein the samplediluent may have a pH of 7.4.

Any of the methods, tests, assays, kits, or systems disclosed herein maycomprise at least one detection reagent configured to selectively bindwith an analyte (for example NF-L, GFAP, UCH L1, or Tau) and supportdetection of a detectable signal (for example through phosphorescence)in an immunoassay. In certain embodiments, for example, the at least onedetection reagent may comprise a first detection reagent, a seconddetection reagent, and a third detection reagent.

In certain embodiments, for example, the first detection reagent maycomprise one or more tagged antibodies (for example, an anti-human NF-Lmouse IgG antibody, a Tau antibody coupled with biotin, an anti-humanUCH L1 mouse IgG antibody and an anti-human GFAP mouse IgG antibody)that specifically binds to an analyte. In certain embodiments, forexample, the first detection reagent may comprise an aqueous solution(for example a solution that may contain 90-100% water) containing theone or more tagged antibodies at a concentration of between 0.01% and1%, between 0.05% and 0.5%, or at a concentration of between 0.01% and0.1% (for example, an anti-human NF-L mouse IgG antibody, a Tau antibodycoupled with biotin, an anti-human UCH L1 mouse IgG antibody and ananti-human GFAP mouse IgG antibody each at a concentration between 0.01%and 0.1%).

In certain embodiments, for example, the first detection reagent maycomprise a plurality (for example 2, 3, 4, 5, 6, 7, 8, 9, 10, or morethan 10) of components selected from the group consisting of one or morebuffers (or a buffering system) (for example phosphate buffer), one ormore ions (for example Na⁺, K⁺ and/or Cl⁻ ions), one or more ionic salts(for example NaCl and KCl), one or more complexing agents (for exampleEDTA disodium salt dehydrate), BSA, one or more blocking agents (forexample interference blocker), and one or more anti-microbial agents(for example a mixture of 2-methyl-3(2H)-isothiazolone and5-chloro-2-methyl-3(2H)-isothiazolone).

In certain embodiments, for example, the first detection reagent maycomprise one or more buffers, or a buffering system (for example one ormore of the buffers or buffering systems disclosed herein or in one ofthe INCORPORATED REFERENCES). In certain embodiments, for example, theone or more buffers may be selected from the group consisting of citratebuffers, phosphate buffers, borate buffers,tris(hydroxymethyl)aminomethane (Tris) buffers, barbital buffers,bicarbonates, as well as Good buffers (non-toxic to cells; not absorbedthrough cell membranes and feature pKa values at or near physiologicalpH) such as but not limited to N,N′-bis(2-hydroxyethyl)glycine (BIC IN),2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-1,3-propanediol(BISTRIS), 2-(cyclohexylamino)ethane-2-sulfonic acid (CHES),N-2-(hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES),N-(2-hydroxyethyl)piperazine-N′-3-propanesulfonic acid (HEPPS),morpholinoethanesulfonic acid (MES), morpholinopropanesulfonic acid(MOPS), piperazine-N, N′-bis(2-ethanesulfonic acid) (PIPES),N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES),N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS), andN-tris(hydroxymethyl)methylglycine (TRICINE), or a combination of two ormore of the foregoing. In certain embodiments, for example, the firstdetection reagent may comprise a buffering system comprising acombination of buffers. In certain embodiments, for example, the RGPreagent may contain a phosphate buffer (for example phosphate at aconcentration of between 10 mM and 100 mM, such as phosphate at aconcentration in the range of between 30 mM and 70 mM, or 50 mM, orsodium phosphate (dibasic) at a concentration of between 0.1% and 30%,such as sodium phosphate (dibasic) at a concentration of between 0.5%and 20%, or at a concentration of between 1% and 10%, and potassiumphosphate (monobasic) at a concentration of between 0.001% and 1%, suchas potassium phosphate (monobasic) at a concentration of between 0.005%and 0.5% or at a concentration of between 0.01% and 0.1%).

In certain embodiments, for example, the first detection reagent maycomprise one or more ions (for example one or more of the ions disclosedherein or in one of the INCORPORATED REFERENCES). In certainembodiments, for example, the one or more ions may be selected from thegroup consisting of Li⁺, Na⁺, K⁺, Mg2⁺, Ca2⁺, Cu2⁺, Mn2⁺, Fe2⁺, Fe3⁺,NH4⁺, Cl⁻, Br⁻, carbonate, hydrogen carbonate, hydrogen sulfate,hydrogen sulfite, sulfate, sulfite, monohydrogen phosphate, dihydrogenphosphate, nitrate, nitrite, permanganate, silicate, sulphates,pyrosulphates, pyrophosphates, citrates, cacodylates and other suitableions. In certain embodiments, for example, the one or more ions maycomprise Cl⁻ (for example Cl⁻ at a concentration of between 100 mM and200 mM, such as Cl⁻ at a concentration in the range of between 120 mMand 160 mM, or 141.7 mM). In certain embodiments, for example, the oneor more ions may comprise Na⁺ (for example Na⁺ at a concentration ofbetween 100 mM and 200 mM, such as Na⁺ at a concentration in the rangeof between 120 mM and 160 mM, or 137 mM). In certain embodiments, forexample, the one or more ions may comprise K⁺ (for example K⁺ at aconcentration of between 1 mM and 5 mM, such as K⁺ at a concentration inthe range of between 2 mM and 3 mM, or 2.7 mM).

In certain embodiments, for example, the ions may form one or more ionicsalts (for example one or more of the ionic salts disclosed herein or inone of the INCORPORATED REFERENCES). In certain embodiments, forexample, the one or more ionic salts may be selected from the groupconsisting of KCl, NaCl, MgCl₂, kh2PO4, K2HPO4, NaH2PO4, Na2HPO4, NaHCO₃and other suitable ionic salts. In certain embodiments, for example, theone or more ionic salts may comprise KCl (for example KCl at aconcentration of between 1 mM and 5 mM, such as KCl at a concentrationin the range of between 2 mM and 3 mM, or 2.7 mM, or KCl at aconcentration of 0.001-1%, such as KCl at a concentration of between0.005% and 0.5%, or at a concentration of between 0.01% and 0.1%). Incertain embodiments, for example, the one or more ionic salts maycomprise NaCl (for example NaCl at a concentration of between 100 mM and200 mM, such as NaCl at a concentration in the range of between 120 mMand 160 mM, or 137 mM, or NaCl at a concentration of between 0.01% and10%, such as NaCl at a concentration of between 0.05% and 5%, or at aconcentration of 0.1% and 1%).

In certain embodiments, for example, the first detection reagent maycomprise the nonionic surfactant polyol sold under the trademarkPluronic™ F-127 (for example Pluronic™ F-127 at a concentration of0.001-1%, such as Pluronic™ F-127 at a concentration of between 0.005%and 0.5%, or at a concentration of between 0.01% and 0.1%).

In certain embodiments, for example, the SBG reagent may comprise one ormore complexing agents that are capable of forming a complex with ametal ion (for example one of the complexing agents disclosed herein orin one of the INCORPORATED REFERENCES). In certain embodiments, forexample, the one or more complexing agents may be selected from thegroup consisting of EDTA, deferoxamine (DESFERAL), NTA,β-alaninediacetic acid (β-ADA), diethylenetriaminepentaacetic acid(DTPA), diethylenetriaminepentakis-methylenephosphonic acid (DTPMP),nitrilotriacetic acid (NTA), N-bis[2-1,2-dicarboxyethoxy)ethyl]glycine(BCAS), N-bis[2-1,2-dicarboxyethoxy)ethyl]aspartic acid (BCA6),N-(hydroxyethyl)-ethylenediaminetriacetic acid (HEDTA), gluconic acidand tetracis(2-hydroxypropyl)ethylenediamine (THPED), other suitablecomplexing agents, or a combination of two or more of the foregoing. Incertain embodiments, for example, the complexing agent may comprise EDTAor a salt of EDTA (for example EDTA or salt of EDTA at a concentrationof between 1 mM and 50 mM, of between 1 mM and 10 mM, or at aconcentration of 5 mM, or EDTA or salt of EDTA at a concentration ofbetween 0.01 and 10%, between 0.05% and 5%, or at a concentration ofbetween 0.1% and 1%). In certain embodiments, for example, the salt ofEDTA may be EDTA disodium salt dihydrate.

In certain embodiments, for example, the first detection reagent maycomprise BSA (for example BSA at a concentration of between 0.1% and30%, such as BSA at a concentration of between 0.5% and 20%, or at aconcentration of between 0.1% and 10%).

In certain embodiments, for example, the first detection reagent maycomprise one or more anti-microbial agents (for example one of theanti-microbial agents disclosed herein or in one of the INCORPORATEDREFERENCES). In certain embodiments, for example, the one or moreanti-microbial agents may be selected from the group consisting ofbenzalkonium chloride, sodium azide, sodium fluoride, phenoxyethanol,sodium dehydroacetate, chlorobutanol, phenylethanol, 4-chloroxylenol,1-hydroxypyridine-2-thione, paraben derivatives, glutaraldehyde,formaldehyde, nalidixic acid, sodium-2-pyridinethiol-1-oxide (sodiumOmadine™), Triadine™ 3, Triadine™ 10, various combinations of5-chloro-2-methyl-4-isothiazolin-2-one, 2-methyl-4-isothiazolin-3-one,5-bromo-5-nitro-1,3-dioxane, derivatives of each of these compounds, ora combination of two or more of the foregoing. In certain embodiments,for example, the one or more anti-microbial agents may be commerciallyavailable and selected from the group consisting of reagents comprisingaqueous combinations of 5-chloro-2-methyl-4-methyl-4-isothiazolin-2-oneand 2-methyl-4-isothiazolin-3-one (Supelco, under the trademarkProclin), for example, Proclin™ 150 reagent (Supelco, an aqueous mixtureof 1.15% of 5-chloro-2-methyl-4-isothiazolin-3-one and 0.35% of2-methyl-4-isothiazolin-3-one), ProClin™ 300 reagent (Supelco, a mixtureof 2.3% of 5-chloro-2-methyl-4-isothiazolin-3-one and 0.7% of2-methyl-4-isothiazolin-3-one in a solvent consisting of a modifiedglycol and alkyl carboxylate), Proclin™ 5000 reagent (Supelco,2-methyl-4-isothiazolin-3-one in a dipropylene glycol solvent),Bronidox® L reagent (Cognis Corporation, biocide5-bromo-5-nitro-1,3-dioxane), or a combination of two or more of theforegoing. In certain embodiments, for example, the first detectionreagent may comprise a combination of two or more anti-microbial agentsin any ratio effective to combat microbial growth. In certainembodiments, for example, the one or more anti-microbial agents maycomprise benzalkonium chloride. In certain embodiments, for example, theone or more anti-microbial agents may comprise ProClin™ 300 reagent (forexample ProClin™ 300 reagent at a concentration of between 0.005% and0.05% (for example 0.02%), between 0.05% and 0.5%, between 0.5% and 1%,between 1% and 5%, or at a concentration of 2%). In certain embodiments,for example, the one or more anti-microbial agents may comprise amixture of 2-methyl-3(2H)-isothiazolone and5-chloro-2-methyl-3(2H)-isothiazolone (for example the mixture at aconcentration of between 0.01% and 10%, between 0.05% and 5%, or at aconcentration of between 0.1% and 1%).

In certain embodiments, for example, the first detection reagent maycomprise one or more blocking agents. In certain embodiments, forexample, the blocking agent may be an interference blocker at aconcentration of between 0.01% and 10%, between 0.05% and 5%, or at aconcentration of between 0.1% and 1%.

In certain embodiments, for example, the first detection reagent maycomprise water, sodium phosphate (dibasic), potassium phosphate(monobasic), NaCl, KCl, EDTA disodium salt dihydrate, BSA, aninterference blocker, an anti-microbial agent (or a mixture ofanti-microbial agents), and one or more first detection reagents (forexample one or more tagged antibodies that each is specific to ananalyte). In certain embodiments, for example, the first detectionreagent may comprise: water at a concentration between 90% and 100%,sodium phosphate (dibasic) at a concentration between 1% to 10%,potassium phosphate (monobasic) at a concentration between 0.01% and0.1%, NaCl at a concentration between 0.1% and 1%, KCl at aconcentration of between 0.01% and 0.1%, EDTA disodium salt dihydrate ata concentration of between 0.1% and 1%, BSA at a concentration ofbetween 1% and 10%, interference blocker at a concentration of between0.1% and 1.0%, an anti-microbial agent at a concentration between 0.1%and 1%, an anti-human NF-L mouse IgG antibody at a concentration between0.01% and 0.1%), a Tau antibody coupled with biotin at a concentrationbetween 0.01% and 0.1%), an anti-human UCH L1 mouse IgG antibody at aconcentration between 0.01% and 0.1%), and an anti-human GFAP mouse IgGantibody at a concentration between 0.01% and 0.1%).

In certain embodiments, for example the second detection reagent maycomprise an enzyme conjugate (for example streptavidin-β-galactosidase(SBG)). In certain embodiments, for example, the second detectionreagent may comprise an aqueous solution (for example a solution thatmay contain 90-100% water). In certain embodiments, for example, thesecond detection reagent may comprise a plurality (for example 2, 3, 4,5, 6, 7, 8, 9, 10, or more than 10) of components selected from thegroup consisting of one or more buffers (or a buffering system) (forexample phosphate buffer), one or more ions (for example Na⁺, K⁺, Mg²⁺and/or Cl⁻ ions), one or more ionic salts (for example NaCl, MgCl₂ andKCl), one or more complexing agents (for exampleethylenediaminetetraacetic acid (EDTA)), BSA, Tween-20, one or moreenzyme conjugates (for example SBG) and one or more anti-microbialagents (for example a mixture of 2-methyl-3(2H)-isothiazolone and5-chloro-2-methyl-3(2H)-isothiazolone).

In certain embodiments, for example, the second detection reagent maycomprise one or more buffers, or a buffering system (for example one ormore of the buffers or buffering systems disclosed herein or in one ofthe INCORPORATED REFERENCES). In certain embodiments, for example, theone or more buffers may be selected from the group consisting of citratebuffers, phosphate buffers, borate buffers,tris(hydroxymethyl)aminomethane (Tris) buffers, barbital buffers,bicarbonates, as well as Good buffers (non-toxic to cells; not absorbedthrough cell membranes and feature pKa values at or near physiologicalpH) such as but not limited to N,N′-bis(2-hydroxyethyl)glycine (BIC IN),2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-1,3-propanediol(BISTRIS), 2-(cyclohexylamino)ethane-2-sulfonic acid (CHES),N-2-(hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES),N-(2-hydroxyethyl)piperazine-N′-3-propanesulfonic acid (HEPPS),morpholinoethanesulfonic acid (MES), morpholinopropanesulfonic acid(MOPS), piperazine-N, N′-bis(2-ethanesulfonic acid) (PIPES),N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES),N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS), andN-tris(hydroxymethyl)methylglycine (TRICINE), or a combination of two ormore of the foregoing. In certain embodiments, for example, the seconddetection reagent may comprise a buffering system comprising acombination of buffers. In certain embodiments, for example, the RGPreagent may contain a phosphate buffer (for example phosphate at aconcentration of between 10 mM and 100 mM, such as phosphate at aconcentration in the range of between 30 mM and 70 mM, or 50 mM, orsodium phosphate (dibasic) at a concentration of between 0.1% and 20%,such as sodium phosphate (dibasic) at a concentration of between 0.5%and 10%, or at a concentration of between 1% and 5%, and potassiumphosphate (monobasic) at a concentration of between 0.05% and 20%, suchas potassium phosphate (monobasic) at a concentration of between 0.1%and 10% or at a concentration of between 0.5% and 2%).

In certain embodiments, for example, the second detection reagent maycomprise one or more ions (for example one or more of the ions disclosedherein or in one of the INCORPORATED REFERENCES). In certainembodiments, for example, the one or more ions may be selected from thegroup consisting of Li⁺, Na⁺, K⁺, Mg2⁺, Ca2⁺, Cu2⁺, Mn2⁺, Fe2⁺, Fe3⁺,NH4⁺, Cl⁻, Br⁻, carbonate, hydrogen carbonate, hydrogen sulfate,hydrogen sulfite, sulfate, sulfite, monohydrogen phosphate, dihydrogenphosphate, nitrate, nitrite, permanganate, silicate, sulphates,pyrosulphates, pyrophosphates, citrates, cacodylates and other suitableions. In certain embodiments, for example, the one or more ions maycomprise Cl⁻ (for example Cl⁻ at a concentration of between 100 mM and200 mM, such as Cl⁻ at a concentration in the range of between 120 mMand 160 mM, or 141.7 mM). In certain embodiments, for example, the oneor more ions may comprise Na⁺ (for example Na⁺ at a concentration ofbetween 100 mM and 200 mM, such as Na⁺ at a concentration in the rangeof between 120 mM and 160 mM, or 137 mM). In certain embodiments, forexample, the one or more ions may comprise K⁺ (for example K⁺ at aconcentration of between 1 mM and 5 mM, such as K⁺ at a concentration inthe range of between 2 mM and 3 mM, or 2.7 mM).

In certain embodiments, for example, the ions may form one or more ionicsalts (for example one or more of the ionic salts disclosed herein or inone of the INCORPORATED REFERENCES). In certain embodiments, forexample, the one or more ionic salts may be selected from the groupconsisting of KCl, NaCl, MgCl₂, KH2PO4, K2HPO4, NaH2PO4, Na2HPO4, NaHCO₃and other suitable ionic salts. In certain embodiments, for example, theone or more ionic salts may comprise KCl (for example KCl at aconcentration of between 1 mM and 5 mM, such as KCl at a concentrationin the range of between 2 mM and 3 mM, or 2.7 mM, or KCl at aconcentration of 0.01-10%, such as KCl at a concentration of between0.05% and 5%, or at a concentration of between 0.1% and 1%). In certainembodiments, for example, the one or more ionic salts may comprise NaCl(for example NaCl at a concentration of between 100 mM and 200 mM, suchas NaCl at a concentration in the range of between 120 mM and 160 mM, or137 mM, or NaCl at a concentration of between 0.01% and 10%, such asNaCl at a concentration of between 0.05% and 5%, or at a concentrationof 0.1% and 1%). In certain embodiments, for example, the one or moreionic salts may comprise MgCl₂ (for example MgCl₂ at a concentration ofbetween 0.1 mM and 5 mM, such as MgCl₂ at a concentration in the rangeof between 0.5 mM and 2.5 mM, or 1 mM, or MgCl₂ at a concentration ofbetween 0.01% and 10%, such as MgCl₂ at a concentration of between 0.05%and 5%, or at a concentration of 0.1% and 1%).

In certain embodiments, for example, the second detection reagent maycomprise one or more complexing agents that are capable of forming acomplex with a metal ion (for example one of the complexing agentsdisclosed herein or in one of the INCORPORATED REFERENCES). In certainembodiments, for example, the one or more complexing agents may beselected from the group consisting of EDTA, deferoxamine (DESFERAL),NTA, β-alaninediacetic acid (β-ADA), diethylenetriaminepentaacetic acid(DTPA), diethylenetriaminepentakis-methylenephosphonic acid (DTPMP),nitrilotriacetic acid (NTA), N-bis[2-1,2-dicarboxyethoxy)ethyl]glycine(BCAS), N-bis[2-1,2-dicarboxyethoxy)ethyl]aspartic acid (BCA6),N-(hydroxyethyl)-ethylenediaminetriacetic acid (HEDTA), gluconic acidand tetracis(2-hydroxypropyl)ethylenediamine (THPED), other suitablecomplexing agents, or a combination of two or more of the foregoing. Incertain embodiments, for example, the complexing agent may comprise EDTAor a salt of EDTA (for example EDTA or salt of EDTA at a concentrationof between 1 mM and 50 mM, of between 1 mM and 10 mM, or at aconcentration of 5 mM, or EDTA or salt of EDTA at a concentration ofbetween 0.01 and 10%, between 0.05% and 5%, or at a concentration ofbetween 0.1% and 1%). In certain embodiments, for example, the salt ofEDTA may be EDTA disodium salt dihydrate.

In certain embodiments, for example, the second detection reagent maycomprise BSA (for example BSA at a concentration of between 0.1% and30%, such as BSA at a concentration of between 0.5% and 20%, or at aconcentration of between 0.1% and 10%).

In certain embodiments, for example, the second detection reagent maycomprise tween 20 (for example tween 20 at a concentration of between0.01% and 10%, such as tween 20 at a concentration of between 0.05% and5%, or at a concentration of between 0.1% and 1%).

In certain embodiments, for example, the second detection reagent maycomprise one or more enzyme conjugates. In certain embodiments, forexample, the enzyme conjugate may be SBG (for example SBG at aconcentration of between 0.001% and 1%, such as SBG at a concentrationof between 0.005% and 0.5%, or at a concentration of between 0.01% and0.1%).

In certain embodiments, for example, the second detection reagent maycomprise one or more anti-microbial agents (for example one of theanti-microbial agents disclosed herein or in one of the INCORPORATEDREFERENCES). In certain embodiments, for example, the one or moreanti-microbial agents may be selected from the group consisting ofbenzalkonium chloride, sodium azide, sodium fluoride, phenoxyethanol,sodium dehydroacetate, chlorobutanol, phenylethanol, 4-chloroxylenol,1-hydroxypyridine-2-thione, paraben derivatives, glutaraldehyde,formaldehyde, nalidixic acid, sodium-2-pyridinethiol-1-oxide (sodiumOmadine™), Triadine™ 3, Triadine™ 10, various combinations of5-chloro-2-methyl-4-isothiazolin-2-one, 2-methyl-4-isothiazolin-3-one,5-bromo-5-nitro-1,3-dioxane, derivatives of each of these compounds, ora combination of two or more of the foregoing. In certain embodiments,for example, the one or more anti-microbial agents may be commerciallyavailable and selected from the group consisting of reagents comprisingaqueous combinations of 5-chloro-2-methyl-4-methyl-4-isothiazolin-2-oneand 2-methyl-4-isothiazolin-3-one (Supelco, under the trademarkProclin), for example, Proclin™ 150 reagent (Supelco, an aqueous mixtureof 1.15% of 5-chloro-2-methyl-4-isothiazolin-3-one and 0.35% of2-methyl-4-isothiazolin-3-one), ProClin™ 300 reagent (Supelco, a mixtureof 2.3% of 5-chloro-2-methyl-4-isothiazolin-3-one and 0.7% of2-methyl-4-isothiazolin-3-one in a solvent consisting of a modifiedglycol and alkyl carboxylate), Proclin™ 5000 reagent (Supelco,2-methyl-4-isothiazolin-3-one in a dipropylene glycol solvent),Bronidox® L reagent (Cognis Corporation, biocide5-bromo-5-nitro-1,3-dioxane), or a combination of two or more of theforegoing. In certain embodiments, for example, the RGP reagent maycomprise a combination of two or more anti-microbial agents in any ratioeffective to combat microbial growth. In certain embodiments, forexample, the one or more anti-microbial agents may comprise benzalkoniumchloride. In certain embodiments, for example, the one or moreanti-microbial agents may comprise ProClin™ 300 reagent (for exampleProClin™ 300 reagent at a concentration of between 0.005% and 0.05% (forexample 0.02%), between 0.05% and 0.5%, between 0.5% and 1%, between 1%and 5%, or at a concentration of 2%). In certain embodiments, forexample, the one or more anti-microbial agents may comprise a mixture of2-methyl-3(2H)-isothiazolone and 5-chloro-2-methyl-3(2H)-isothiazolone(for example the mixture at a concentration of between 0.01% and 5%,between 0.05% and 2%, or at a concentration of between 0.1% and 1%).

In certain embodiments, for example, the second detection reagent maycomprise water, sodium phosphate (dibasic), potassium phosphate(monobasic), NaCl, KCl, EDTA disodium salt dihydrate, BSA, Tween 20,MgCl₂, an enzyme conjugate and an anti-microbial agent (or a mixture ofanti-microbial agents). In certain embodiments, for example, the seconddetection reagent may comprise: water at a concentration between 90% and100%, sodium phosphate (dibasic) at a concentration between 1% and 5%,potassium phosphate (monobasic) at a concentration between 0.5% and 2%,NaCl at a concentration between 0.1% and 1%, KCl at a concentration ofbetween 0.1% and 1%, MgCl₂ at a concentration between 0.1% and 1%, EDTAdisodium salt dihydrate at a concentration between 0.1% and 1%, BSA atthe concentration of between 1% and 10%, tween 20 at a concentration ofbetween 0.1% and 1%, an enzyme conjugate at a concentration of between0.01% and 0.1%, and an anti-microbial agent at a concentration between0.1% and 1%.

In certain embodiments, for example the third detection reagent maycomprise a fluorogenic enzyme substrate (for example resorufinb-galactopyranoside (RGP)). In certain embodiments, for example, thethird detection reagent may comprise aqueous solution (for example asolution that may contain 90-100% water). In certain embodiments, forexample, the third detection reagent may comprise a plurality (forexample 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10) of componentsselected from the group consisting of one or more buffers (or abuffering system) (for example phosphate buffer), one or more ions (forexample Na⁺, K⁺ and/or Cl− ions), one or more ionic salts (for exampleNaCl and KCl), Pluronic™ F-127, resorufin b-galactopyranoside, and oneor more anti-microbial agents (for example a mixture of2-methyl-3(2H)-isothiazolone and 5-chloro-2-methyl-3(2H)-isothiazolone).

In certain embodiments, for example, the third detection reagent maycomprise one or more buffers, or a buffering system (for example one ormore of the buffers or buffering systems disclosed herein or in one ofthe INCORPORATED REFERENCES). In certain embodiments, for example, theone or more buffers may be selected from the group consisting of citratebuffers, phosphate buffers, borate buffers,tris(hydroxymethyl)aminomethane (Tris) buffers, barbital buffers,bicarbonates, as well as Good buffers (non-toxic to cells; not absorbedthrough cell membranes and feature pKa values at or near physiologicalpH) such as but not limited to N,N′-bis(2-hydroxyethyl)glycine (BIC IN),2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-1,3-propanediol(BISTRIS), 2-(cyclohexylamino)ethane-2-sulfonic acid (CHES),N-2-(hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES),N-(2-hydroxyethyl)piperazine-N′-3-propanesulfonic acid (HEPPS),morpholinoethanesulfonic acid (MES), morpholinopropanesulfonic acid(MOPS), piperazine-N, N′-bis(2-ethanesulfonic acid) (PIPES),N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES),N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS), andN-tris(hydroxymethyl)methylglycine (TRICINE), or a combination of two ormore of the foregoing. In certain embodiments, for example, the thirddetection reagent may comprise a buffering system comprising acombination of buffers. In certain embodiments, for example, the thirddetection reagent may contain a phosphate buffer (for example phosphateat a concentration of between 10 mM and 100 mM, such as phosphate at aconcentration in the range of between 30 mM and 70 mM, or 50 mM, orsodium phosphate (dibasic) at a concentration of between 0.01% and 5%,such as sodium phosphate (dibasic) at a concentration of between 0.05%and 1%, or at a concentration of between 0.1% and 0.5%, and potassiumphosphate (monobasic) at a concentration of between 0.001% and 1%, suchas potassium phosphate (monobasic) at a concentration of between 0.005%and 0.5% or at a concentration of between 0.01% and 0.1%).

In certain embodiments, for example, the third detection reagent maycomprise one or more ions (for example one or more of the ions disclosedherein or in one of the INCORPORATED REFERENCES). In certainembodiments, for example, the one or more ions may be selected from thegroup consisting of Li⁺, Na⁺, K⁺, Mg2⁺, Ca2⁺, Cu2⁺, Mn2⁺, Fe2⁺, Fe3⁺,NH4⁺, Cl⁻, Br⁻, carbonate, hydrogen carbonate, hydrogen sulfate,hydrogen sulfite, sulfate, sulfite, monohydrogen phosphate, dihydrogenphosphate, nitrate, nitrite, permanganate, silicate, sulphates,pyrosulphates, pyrophosphates, citrates, cacodylates and other suitableions. In certain embodiments, for example, the one or more ions maycomprise Cl⁻ (for example Cl⁻ at a concentration of between 100 mM and200 mM, such as Cl⁻ at a concentration in the range of between 120 mMand 160 mM, or 141.7 mM). In certain embodiments, for example, the oneor more ions may comprise Na⁺ (for example Na⁺ at a concentration ofbetween 100 mM and 200 mM, such as Na⁺ at a concentration in the rangeof between 120 mM and 160 mM, or 137 mM). In certain embodiments, forexample, the one or more ions may comprise K⁺ (for example K⁺ at aconcentration of between 1 mM and 5 mM, such as K⁺ at a concentration inthe range of between 2 mM and 3 mM, or 2.7 mM)

In certain embodiments, for example, the ions may form one or more ionicsalts (for example one or more of the ionic salts disclosed herein or inone of the INCORPORATED REFERENCES). In certain embodiments, forexample, the one or more ionic salts may be selected from the groupconsisting of KCl, NaCl, MgCl₂, KH2PO4, K2HPO4, NaH2PO4, Na2HPO4, NaHCO₃and other suitable ionic salts. In certain embodiments, for example, theone or more ionic salts may comprise KCl (for example KCl at aconcentration of between 1 mM and 5 mM, such as KCl at a concentrationin the range of between 2 mM and 3 mM, or 2.7 mM, or KCl at aconcentration of 0.001-1%, such as KCl at a concentration of between0.005% and 0.5%, or at a concentration of between 0.01% and 0.1%). Incertain embodiments, for example, the one or more ionic salts maycomprise NaCl (for example NaCl at a concentration of between 100 mM and200 mM, such as NaCl at a concentration in the range of between 120 mMand 160 mM, or 137 mM, or NaCl at a concentration of between 0.01% and10%, such as NaCl at a concentration of between 0.05% and 5%, or at aconcentration of 0.1% and 1%).

In certain embodiments, for example, the third detection reagent maycomprise Pluronic™ F-127 (for example Pluronic™ F-127 at a concentrationof 0.001-1%, such as Pluronic™ F-127 at a concentration of between0.005% and 0.5%, or at a concentration of between 0.01% and 0.1%).

In certain embodiments, for example, the third detection reagent maycomprise resorufin b-galactopyranoside (for example resorufinb-galactopyranoside at a concentration of 0.0001-0.1%, such as resorufinb-galactopyranoside at a concentration of between 0.0005% and 0.05%, orat a concentration of between 0.001% and 0.01%).

In certain embodiments, for example, the third detection reagent maycomprise one or more anti-microbial agents (for example one of theanti-microbial agents disclosed herein or in one of the INCORPORATEDREFERENCES). In certain embodiments, for example, the one or moreanti-microbial agents may be selected from the group consisting ofbenzalkonium chloride, sodium azide, sodium fluoride, phenoxyethanol,sodium dehydroacetate, chlorobutanol, phenylethanol, 4-chloroxylenol,1-hydroxypyridine-2-thione, paraben derivatives, glutaraldehyde,formaldehyde, nalidixic acid, sodium-2-pyridinethiol-1-oxide (sodiumOmadine™), Triadine™ 3, Triadine™ 10, various combinations of5-chloro-2-methyl-4-isothiazolin-2-one, 2-methyl-4-isothiazolin-3-one,5-bromo-5-nitro-1,3-dioxane, derivatives of each of these compounds, ora combination of two or more of the foregoing. In certain embodiments,for example, the one or more anti-microbial agents may be commerciallyavailable and selected from the group consisting of reagents comprisingaqueous combinations of 5-chloro-2-methyl-4-methyl-4-isothiazolin-2-oneand 2-methyl-4-isothiazolin-3-one (Supelco, under the trademarkProclin), for example, Proclin™ 150 reagent (Supelco, an aqueous mixtureof 1.15% of 5-chloro-2-methyl-4-isothiazolin-3-one and 0.35% of2-methyl-4-isothiazolin-3-one), ProClin™ 300 reagent (Supelco, a mixtureof 2.3% of 5-chloro-2-methyl-4-isothiazolin-3-one and 0.7% of2-methyl-4-isothiazolin-3-one in a solvent consisting of a modifiedglycol and alkyl carboxylate), Proclin™ 5000 reagent (Supelco,2-methyl-4-isothiazolin-3-one in a dipropylene glycol solvent),Bronidox® L reagent (Cognis Corporation, biocide5-bromo-5-nitro-1,3-dioxane), or a combination of two or more of theforegoing. In certain embodiments, for example, the third detectionreagent may comprise a combination of two or more anti-microbial agentsin any ratio effective to combat microbial growth. In certainembodiments, for example, the one or more anti-microbial agents maycomprise benzalkonium chloride. In certain embodiments, for example, theone or more anti-microbial agents may comprise ProClin™ 300 reagent (forexample ProClin™ 300 reagent at a concentration of between 0.005% and0.05% (for example 0.02%), between 0.05% and 0.5%, between 0.5% and 1%,between 1% and 5%, or at a concentration of 2%). In certain embodiments,for example, the one or more anti-microbial agents may comprise amixture of 2-methyl-3(2H)-isothiazolone and5-chloro-2-methyl-3(2H)-isothiazolone (for example the mixture at aconcentration of between 0.01% and 5%, between 0.05% and 1%, or at aconcentration of between 0.1% and 0.5%).

In certain embodiments, for example, the third detection reagent maycomprise water, sodium phosphate (dibasic), potassium phosphate(monobasic), NaCl, KCl, Pluronic™ F-127, resorufin b-galactopyranosideand an anti-microbial agent (or a mixture of anti-microbial agents). Incertain embodiments, for example, the third detection reagent maycomprise: water at a concentration between 90% and 100%, sodiumphosphate (dibasic) at a concentration between 0.1% to 0.5%, potassiumphosphate (monobasic) at a concentration between 0.01% and 0.1%, NaCl ata concentration between 0.1% and 1%, KCl at a concentration of between0.01% and 0.1%, Pluronic™ F-127 at a concentration of between 0.01% and0.1%, resorufin b-galactopyranoside at a concentration of between 0.001%and 0.01%, and an anti-microbial agent at a concentration between 0.1%and 0.5%.

Certain embodiments may provide, for example, methods, tests, assays(for example digital immunoassays), kits, or systems to quantifyabnormal levels of one or more biomarkers present in a sample ofphysiological fluid taken from a subject. In certain embodiments, forexample, the physiological fluid may be plasma or serum obtained from ablood sample (for example a venous blood sample). In certainembodiments, for example, the physiological fluid may be taken from asubject following an event (for example an event comprising a medicalprocedure, or a potentially neurological condition-inducing event). Incertain embodiments, for example, the physiological fluid may be takenwithin 1 week following the event, for example within 36 hours followingthe event, within 24 hours, within 12 hours, within 11 hours, within 10hours, within 9 hours, within 8 hours, within 7 hours, within 6 hours,within 5 hours, within 4 hours, within 3 hours, within 2 hours, within 1hour, within 45 minutes, within 30 minutes, within 15 minutes, within 10minutes, within 5 minutes, or the physiological fluid may be takenwithin 1 minute following the event. In certain embodiments, forexample, the physiological fluid may be taken after at least 10 minutesfollowing the event, for example at least 30 minutes following theevent, at least 1 hour, at least 2 hours, at least 6 hours, at least 8hours, at least 12 hours, at least 24 hours, at least 3 days, or thephysiological fluid may be taken between after at least 7 days followingthe event. In certain embodiments, for example, the physiological fluidmay be taken between 1 hour and 15 days following the event, for examplebetween 1 hour and 2 days following the event, between 1 hour and 12hours, between 6 hours and 3 days, or the physiological fluid may betaken between 6 hours and 10 days following the event.

In certain embodiments, for example, the methods, tests, assays, kits,or systems may be limited to testing a single liquid sample derived fromthe sample of physiological fluid. In certain embodiments, for example,the methods, tests, assays, kits, or systems may comprise testingmultiple liquid samples derived from multiple samples of physiologicalfluid (for example samples taken from the subject at spaced timeintervals). In certain embodiments, for example, multiple samples ofphysiological fluid may be taken from a subject at different timesduring a predetermined time window. In certain embodiments, for example,between 2 and 10 physiological fluid samples (for example between 3 and5 physiological fluid samples) may be taken from the subject during atime window of less than 20 days, for example during a time window ofless than 10 days, less than 7 days, less than 100 hours, less than 48hours, or less than 24 hours.

In certain embodiments, for example, the sample of physiological fluidmay weigh less than 5 gram, for example less than 2 grams, less than 1gram, less than 0.5 grams, less than 0.25 grams, less than 0.1 grams,less than 0.01 grams, less than 1 mg, less than 100 mcg, less than 10mcg, less than 1 mcg, less than 0.1 mcg, or the sample of physiologicalfluid may weigh less than 0.01 mcg.

In certain embodiments, for example, the sample of physiological fluidmay be maintained at −80° C., may be maintained (for example stored) ata temperature of between −80° C. and −60° C., for example at atemperature of between −60° C. and −40° C., between −40° C. and −20° C.,−20° C. and 0° C., or the sample of physiological fluid may bemaintained at a temperature of between 0° C. and 25° C. In certainembodiments, for example, the sample of physiological fluid may bemaintained (for example stored) under ambient conditions (for exampleambient temperature and/or humidity). In certain embodiments, forexample, the sample of physiological fluid may be maintained at arelative humidity of less than 50%. In certain embodiments, for example,the sample of physiological fluid may be stored for at least 1 month,for example for at least 2 months, at least 3 months, at least 6 months,at least 1 year, at least 2 years, between 1 month and 5 years, between1 month and 3 months, between 1 month and 1 year, between 1 month and 2years, between 1 month and 3 years, or between 1 month and 4 years. Incertain embodiments, for example, the sample of physiological fluid maybe stored for at least 4 years, for example at least 5 years or at least10 years.

In certain embodiments, for example, the methods, tests, assays, kits,or systems may comprise contacting the sample of physiological fluidwith a solution containing assay microbeads (for example the contactingmay occur in a reaction vessel). In certain embodiments, for example,the solution contacted with the sample of physiological fluid may beincubated for a period of time (for example for at least 30 minutes, atleast 1 hour, at least 2 hours, at least 6 hours, at least 12 hours, atleast 24 hours, at least 48 hours, between 1 hour and 2 hours, between 1hour and 6 hours, between 1 hour and 12 hours, or between 12 hours and48 hours.

Certain embodiments may provide, for example, methods, tests, assays,kits, or systems for testing a liquid sample derived from a sample ofphysiological fluid (for example to detect a neurological condition). Incertain embodiments, for example, the sample of physiological fluid maybe at least 1 week old prior to deriving the liquid sample from thesample of physiological fluid, for example at least 2 weeks old, atleast 1 month old, at least 2 months old, at least 3 months old, atleast 6 months old, at least 12 months old, at least 2 years old, atleast 3 years old, at least 4 years old, at least 5 years old, or thesample of physiological fluid may be at least 10 years old prior toderiving the liquid sample from the sample of physiological fluid. Incertain embodiments, for example, the sample of physiological fluid maybe between 1 month and 20 years old prior to deriving the liquid sample,for example between 1 month and 6 years old, between 2 months and 3years old, or the sample of physiological fluid may be between 3 yearsand 10 years old prior to deriving the liquid sample.

In certain embodiments, for example, the methods, tests, assays, kits,or systems may comprise obtaining or quantifying at least one parameterfor at least one biomarker in the liquid sample. In certain embodiments,for example, the at least one biomarker may be a protein biomarker. Incertain embodiments, for example, the at least one biomarker may be aphosphorylated protein biomarker. In certain embodiments, for example,the at least one biomarker may comprise a neurofilament. In certainembodiments, for example, the neurofilament may be a type IVintermediate filaments or heteropolymers. In certain embodiments, forexample, the neurofilament may be selected from the group consisting ofneurofilament heavy chain (NF-H), neurofilament medium chain (NF-M),NF-L, α-internexin, and peripherin. In certain embodiments, for example,the at least one biomarker may comprise NF-L. In certain embodiments,for example, the at least one biomarker may comprise GFAP. In certainembodiments, for example, the at least one biomarker may comprise UCHL1. In certain embodiments, for example, the at least one biomarker maycomprise a tau protein (Tau), for example one isoform of tau protein, 2isoforms of tau protein, 3 isoforms of tau protein, 4 isoforms of tauprotein, 5 isoforms of tau protein, 6 isoforms of tau protein, or aphosphorylated tau protein (for example p-tau-81 or p-tau-231). Incertain embodiments, for example, the tau protein may be tau 23. Incertain embodiments, for example, the tau protein may be tau 24. Incertain embodiments, for example, the tau protein may be tau 37. Incertain embodiments, for example, the tau protein may be tau 34. Incertain embodiments, for example, the tau protein may be tau 39. Incertain embodiments, for example, the tau protein may be tau 40. Incertain embodiments, for example, the tau protein may be phosphorylated.In certain embodiments, for example, the at least one biomarker maycomprise a protein derived from amyloid beta precursor protein (forexample a proteolytic product of amyloid beta precursor protein). Incertain embodiments, for example, the at least one biomarker maycomprise Amyloid beta 40 (A beta 40). In certain embodiments, forexample, the at least one biomarker may comprise Amyloid beta 42 (A beta42). In certain embodiments, for example, the at least one biomarker maycomprise S100 calcium-binding protein B (S100B). In certain embodiments,for example, the at least one biomarker may comprise Neuron-specificenolase (NSE). In certain embodiments, for example, the at least onebiomarker may comprise β-site aPP-cleaving enzyme 1 (BACe1). In certainembodiments, for example, the at least one biomarker may comprise myelinbasic protein (MBP). In certain embodiments, for example, the at leastone biomarker may comprise growth associated protein 43. In certainembodiments, for example, the at least one biomarker may compriseglutamine synthetase. In certain embodiments, for example, the at leastone biomarker may comprise a glycine transporter (for example at leastone of GLYT1 and GLYT2). In certain embodiments, for example, the atleast one biomarker may comprise a neuron specific glycoprotein (forexample GP50). In certain embodiments, for example, the at least onebiomarker may comprise calpain. In certain embodiments, for example, theat least one biomarker may comprise heat shock protein 72. In certainembodiments, for example, the at least one biomarker may comprise abeta-amyloid precursor protein. In certain embodiments, for example, theat least one biomarker may comprise calbindin D-28K. In certainembodiments, for example, the at least one biomarker may comprise aproteolipid protein. In certain embodiments, for example, the at leastone biomarker may comprise a myelin associated glycoprotein. In certainembodiments, for example, the at least one biomarker may comprise acreatine kinase protein (for example CK-BB). In certain embodiments, forexample, the at least one biomarker may comprise an endothelium membraneprotein (for example thrombomodulin).

In certain embodiments, for example, the at least one parameter may bedetermined from measurements for a selected number and combination ofbiomarkers associated with a neurological condition. In certainembodiments, for example, the measurements may comprise concentrationsof at least 2 biomarkers (for example 2 biomarkers, 3 biomarkers, 4biomarkers, 5 biomarkers, 6 biomarkers, 7 biomarkers, 8 biomarkers, 9biomarkers, 10 biomarkers, or more than 10 biomarkers). In certainembodiments, for example, the at least 2 biomarkers may be selected fromthe group consisting of NF-L, GFAP, UCH L1, and Tau. In certainembodiments, for example, the measurements may comprise concentrationsof at least 3 biomarkers. In certain embodiments, for example, the atleast 3 biomarkers may be selected from the group consisting of NF-L,GFAP, UCH L1, and Tau. In certain embodiments, for example, themeasurements may comprise concentrations of at least 4 biomarkers. Incertain embodiments, for example, the at least 4 biomarkers may beselected from the group consisting of NF-L, GFAP, UCH L1, and Tau. Incertain embodiments, for example, the measurements may compriseconcentrations of at least 5 biomarkers. In certain embodiments, forexample, the at least 5 biomarkers may be selected from the groupconsisting of NF-L, GFAP, UCH L1, and Tau. In certain embodiments, forexample, the measurements may comprise concentrations of at least 6biomarkers. In certain embodiments, for example, the at least 2biomarkers may be selected from the group consisting of NF-L, GFAP, UCHL1, and Tau. In certain embodiments, for example, the measurements maycomprise a concentration of NF-L and a concentration of at least oneadditional biomarker (for example concentrations of 2 additionalbiomarkers, 3 additional biomarkers, 4 additional biomarkers, 5additional biomarkers, 6 additional biomarkers, 7 additional biomarkers,8 additional biomarkers, 9 additional biomarkers, 10 additionalbiomarkers, or more than 10 additional biomarkers) selected from thegroup consisting of GFAP, UCH L1, and Tau. In certain embodiments, forexample, the measurements may comprise concentrations of from 2 to 10biomarkers, for example concentrations of from 2 to 8 biomarkers, from 2to 5 biomarkers, or the measurements may comprise concentrations of from2 to 4 biomarkers.

In certain embodiments, for example, the at least one biomarkerindicative of the neurological condition may be indicative of theneurological condition at a concentration in the liquid sample of lessthan 1000 pg/mL, for example less than 100 pg/mL, less than 10 pg/mL,less than 1 pg/mL, less than 0.1 pg/mL, less than 0.01 pg/mL, or the atleast one biomarker may be indicative of the neurological condition at amolar concentration in the liquid sample of less than 0.001 pg/mL. Incertain embodiments, for example, the at least one biomarker may beindicative of the neurological condition at a concentration in theliquid sample of between 0.001 pg/mL and 0.1 pg/mL. In certainembodiments, for example, the at least one biomarker may be indicativeof the neurological condition at a concentration in the liquid sample ofbetween 0.1 pg/mL and 10 pg/mL. In certain embodiments, for example, theat least one biomarker may be indicative of the neurological conditionat a concentration in the liquid sample of between 1 pg/mL and 100pg/mL. In certain embodiments, for example, the at least one biomarkermay be indicative of the neurological condition at a concentration inthe liquid sample of between 10 pg/mL and 1000 pg/mL.

In certain embodiments, for example, NF-L may be indicative of theneurological condition at a concentration in the liquid sample of lessthan 1000 pg/mL, for example less than 100 pg/mL, less than 10 pg/mL,less than 1 pg/mL, less than 0.1 pg/mL, less than 0.01 pg/mL, or NF-Lmay be indicative of the neurological condition at a molar concentrationin the liquid sample of less than 0.001 pg/mL. In certain embodiments,for example, NF-L may be indicative of the neurological condition at aconcentration in the liquid sample of between 0.001 pg/mL and 0.1 pg/mL.In certain embodiments, for example, NF-L may be indicative of theneurological condition at a concentration in the liquid sample ofbetween 0.1 pg/mL and 10 pg/mL. In certain embodiments, for example,NF-L may be indicative of the neurological condition at a concentrationin the liquid sample of between 1 pg/mL and 100 pg/mL. In certainembodiments, for example, NF-L may be indicative of the neurologicalcondition at a concentration in the liquid sample of between 10 pg/mLand 1000 pg/mL.

In certain embodiments, for example, GFAP may be indicative of aneurological condition at a concentration in the liquid sample of lessthan 1000 pg/mL, for example less than 100 pg/mL, less than 10 pg/mL,less than 1 pg/mL, less than 0.1 pg/mL, less than 0.01 pg/mL, or GFAPmay be indicative of the neurological condition at a molar concentrationin the liquid sample of less than 0.001 pg/mL. In certain embodiments,for example, GFAP may be indicative of the neurological condition at aconcentration in the liquid sample of between 0.001 pg/mL and 0.1 pg/mL.In certain embodiments, for example, GFAP may be indicative of theneurological condition at a concentration in the liquid sample ofbetween 0.1 pg/mL and 10 pg/mL. In certain embodiments, for example,GFAP may be indicative of the neurological condition at a concentrationin the liquid sample of between 1 pg/mL and 100 pg/mL. In certainembodiments, for example, GFAP may be indicative of the neurologicalcondition at a concentration in the liquid sample of between 10 pg/mLand 1000 pg/mL.

In certain embodiments, for example, UCH L1 may be indicative of theneurological condition at a concentration in the liquid sample of lessthan 1000 pg/mL, for example less than 100 pg/mL, less than 10 pg/mL,less than 1 pg/mL, less than 0.1 pg/mL, less than 0.01 pg/mL, or UCH L1may be indicative of the neurological condition at a molar concentrationin the liquid sample of less than 0.001 pg/mL. In certain embodiments,for example, UCH L1 may be indicative of the neurological condition at aconcentration in the liquid sample of between 0.001 pg/mL and 0.1 pg/mL.In certain embodiments, for example, UCH L1 may be indicative of theneurological condition at a concentration in the liquid sample ofbetween 0.1 pg/mL and 10 pg/mL. In certain embodiments, for example, UCHL1 may be indicative of the neurological condition at a concentration inthe liquid sample of between 1 pg/mL and 100 pg/mL. In certainembodiments, for example, UCH L1 may be indicative of the neurologicalcondition at a concentration in the liquid sample of between 10 pg/mLand 1000 pg/mL.

In certain embodiments, for example, tau protein may be indicative ofthe neurological condition at a concentration in the liquid sample ofless than 1000 pg/mL, for example less than 100 pg/mL, less than 10pg/mL, less than 1 pg/mL, less than 0.1 pg/mL, less than 0.01 pg/mL, ortau protein may be indicative of the neurological condition at a molarconcentration in the liquid sample of less than 0.001 pg/mL. In certainembodiments, for example, tau protein may be indicative of theneurological condition at a concentration in the liquid sample ofbetween 0.001 pg/mL and 0.1 pg/mL. In certain embodiments, for example,tau protein may be indicative of the neurological condition at aconcentration in the liquid sample of between 0.1 pg/mL and 10 pg/mL. Incertain embodiments, for example, tau protein may be indicative of theneurological condition at a concentration in the liquid sample ofbetween 1 pg/mL and 100 pg/mL. In certain embodiments, for example, tauprotein may be indicative of the neurological condition at aconcentration in the liquid sample of between 10 pg/mL and 1000 pg/mL.

In certain embodiments, for example, A beta 40 may be indicative of theneurological condition at a concentration in the liquid sample of lessthan 1000 pg/mL, for example less than 100 pg/mL, less than 10 pg/mL,less than 1 pg/mL, less than 0.1 pg/mL, less than 0.01 pg/mL, or A beta40 may be indicative of the neurological condition at a molarconcentration in the liquid sample of less than 0.001 pg/mL. In certainembodiments, for example, A beta 40 may be indicative of theneurological condition at a concentration in the liquid sample ofbetween 0.001 pg/mL and 0.1 pg/mL. In certain embodiments, for example,A beta 40 may be indicative of the neurological condition at aconcentration in the liquid sample of between 0.1 pg/mL and 10 pg/mL. Incertain embodiments, for example, A beta 40 may be indicative of theneurological condition at a concentration in the liquid sample ofbetween 1 pg/mL and 100 pg/mL. In certain embodiments, for example, Abeta 40 may be indicative of the neurological condition at aconcentration in the liquid sample of between 10 pg/mL and 1000 pg/mL.

In certain embodiments, for example, A beta 42 may be indicative of theneurological condition at a concentration in the liquid sample of lessthan 1000 pg/mL, for example less than 100 pg/mL, less than 10 pg/mL,less than 1 pg/mL, less than 0.1 pg/mL, less than 0.01 pg/mL, or A beta42 may be indicative of the neurological condition at a molarconcentration in the liquid sample of less than 0.001 pg/mL. In certainembodiments, for example, A beta 42 may be indicative of theneurological condition at a concentration in the liquid sample ofbetween 0.001 pg/mL and 0.1 pg/mL. In certain embodiments, for example,A beta 42 may be indicative of the neurological condition at aconcentration in the liquid sample of between 0.1 pg/mL and 10 pg/mL. Incertain embodiments, for example, A beta 42 may be indicative of theneurological condition at a concentration in the liquid sample ofbetween 1 pg/mL and 100 pg/mL. In certain embodiments, for example, Abeta 42 may be indicative of the neurological condition at aconcentration in the liquid sample of between 10 pg/mL and 1000 pg/mL.

In certain embodiments, for example, S100B may be indicative of theneurological condition at a concentration in the liquid sample of lessthan 1000 pg/mL, for example less than 100 pg/mL, less than 10 pg/mL,less than 1 pg/mL, less than 0.1 pg/mL, less than 0.01 pg/mL, or S100Bmay be indicative of the neurological condition at a molar concentrationin the liquid sample of less than 0.001 pg/mL. In certain embodiments,for example, S100B may be indicative of the neurological condition at aconcentration in the liquid sample of between 0.001 pg/mL and 0.1 pg/mL.In certain embodiments, for example, S100B may be indicative of theneurological condition at a concentration in the liquid sample ofbetween 0.1 pg/mL and 10 pg/mL. In certain embodiments, for example,S100B may be indicative of the neurological condition at a concentrationin the liquid sample of between 1 pg/mL and 100 pg/mL. In certainembodiments, for example, S100B may be indicative of the neurologicalcondition at a concentration in the liquid sample of between 10 pg/mLand 1000 pg/mL.

In certain embodiments, for example, NSE may be indicative of theneurological condition at a concentration in the liquid sample of lessthan 1000 pg/mL, for example less than 100 pg/mL, less than 10 pg/mL,less than 1 pg/mL, less than 0.1 pg/mL, less than 0.01 pg/mL, or NSE maybe indicative of the neurological condition at a molar concentration inthe liquid sample of less than 0.001 pg/mL. In certain embodiments, forexample, NSE may be indicative of the neurological condition at aconcentration in the liquid sample of between 0.001 pg/mL and 0.1 pg/mL.In certain embodiments, for example, NSE may be indicative of theneurological condition at a concentration in the liquid sample ofbetween 0.1 pg/mL and 10 pg/mL. In certain embodiments, for example, NSEmay be indicative of the neurological condition at a concentration inthe liquid sample of between 1 pg/mL and 100 pg/mL. In certainembodiments, for example, NSE may be indicative of the neurologicalcondition at a concentration in the liquid sample of between 10 pg/mLand 1000 pg/mL.

In certain embodiments, for example, the methods, tests, assays, kits,or systems may require a LOQ of no greater than 0.01 pg/mL (for exampleno greater than 0.1 pg/mL, no greater than 0.5 pg/mL, no greater than 1pg/mL, no greater than 2 pg/mL, no greater than 5 pg/mL, no greater than10 pg/mL, or no greater than 50 pg/mL) for tau protein.

In certain embodiments, for example, the methods, tests, assays, kits,or systems may require a LOQ of no greater than 0.01 pg/mL (for exampleno greater than 0.1 pg/mL, no greater than 0.5 pg/mL, no greater than 1pg/mL, no greater than 2 pg/mL, no greater than 5 pg/mL, no greater than10 pg/mL, or no greater than 50 pg/mL) for NF-L.

In certain embodiments, for example, the methods, tests, assays, kits,or systems may require a LOQ of no greater than 0.01 pg/mL (for exampleno greater than 0.1 pg/mL, no greater than 0.5 pg/mL, no greater than 1pg/mL, no greater than 2 pg/mL, no greater than 5 pg/mL, no greater than10 pg/mL, or no greater than 50 pg/mL) for GFAP.

In certain embodiments, for example, the methods, tests, assays, kits,or systems may require a LOQ of no greater than 0.01 pg/mL (for exampleno greater than 0.1 pg/mL, no greater than 0.5 pg/mL, no greater than 1pg/mL, no greater than 2 pg/mL, no greater than 5 pg/mL, no greater than10 pg/mL, or no greater than 50 pg/mL) for UCH L1.

In certain embodiments, for example, the methods, tests, assays, kits,or systems may require a LOQ of no greater than 0.01 pg/mL (for exampleno greater than 0.1 pg/mL, no greater than 0.5 pg/mL, no greater than 1pg/mL, no greater than 2 pg/mL, no greater than 5 pg/mL, no greater than10 pg/mL, or no greater than 50 pg/mL) for A beta 40.

In certain embodiments, for example, the methods, tests, assays, kits,or systems may require a LOQ of no greater than 0.01 pg/mL (for exampleno greater than 0.1 pg/mL, no greater than 0.5 pg/mL, no greater than 1pg/mL, no greater than 2 pg/mL, no greater than 5 pg/mL, no greater than10 pg/mL, or no greater than 50 pg/mL) for A beta 42.

In certain embodiments, for example, the methods, tests, assays, kits,or systems may require a LOQ of no greater than 0.01 pg/mL (for exampleno greater than 0.1 pg/mL, no greater than 0.5 pg/mL, no greater than 1pg/mL, no greater than 2 pg/mL, no greater than 5 pg/mL, no greater than10 pg/mL, or no greater than 50 pg/mL) for S100B.

In certain embodiments, for example, the methods, tests, assays, kits,or systems may require a LOQ of no greater than 0.01 pg/mL (for exampleno greater than 0.1 pg/mL, no greater than 0.5 pg/mL, no greater than 1pg/mL, no greater than 2 pg/mL, no greater than 5 pg/mL, no greater than10 pg/mL, or no greater than 50 pg/mL) for NSE.

In certain embodiments, for example, the at least one biomarker (or anelevated or reduced concentration of the at least one biomarker) may beindicative of a neurological condition occurring during a time ofbetween 1 second and 5 years prior to a date and time on which thephysiological fluid was taken from a subject, for example during a timeof between 1 second and 2 years, at time of between 1 second and 1 year,a time of between 1 second and 6 months, a time of between 1 second and3 months, a time of between 1 second and 1 month, a time of between 1second and 10 days, or the at least one biomarker may be indicative of aneurological condition occurring during a time of between 1 second and 1days prior to a date and time on which the physiological fluid was takenfrom a subject. In certain embodiments, for example, the at least onebiomarker may be indicative of a neurological condition being presentduring a time of between 1 second and 5 years prior to a date and timeon which the physiological fluid was taken from a subject, for exampleduring a time of between 1 second and 2 years, at time of between 1second and 1 year, a time of between 1 second and 6 months, a time ofbetween 1 second and 3 months, a time of between 1 second and 1 month, atime of between 1 second and 10 days, or the at least one biomarker maybe indicative of a neurological condition being present during a time ofbetween 1 second and 1 days prior to a date and time on which thephysiological fluid was taken from a subject.

In certain embodiments, for example, an increased concentration of NF-Lmay be indicative of a neurological condition occurring during a time ofbetween 1 second and 5 years prior to a date and time on which thephysiological fluid was taken from a subject, for example during a timeof between 1 second and 2 years, at time of between 1 second and 1 year,a time of between 1 second and 6 months, a time of between 1 second and3 months, a time of between 1 second and 1 month, a time of between 1second and 10 days, or an increased concentration of NF-L may beindicative of a neurological condition occurring during a time ofbetween 1 second and 1 days prior to a date and time on which thephysiological fluid was taken from a subject. In certain embodiments,for example, an increased concentration of NF-L may be indicative of aneurological condition being present during a time of between 1 secondand 5 years prior to a date and time on which the physiological fluidwas taken from a subject, for example during a time of between 1 secondand 2 years, at time of between 1 second and 1 year, a time of between 1second and 6 months, a time of between 1 second and 3 months, a time ofbetween 1 second and 1 month, a time of between 1 second and 10 days, oran increased concentration of NF-L may be indicative of a neurologicalcondition being present during a time of between 1 second and 1 daysprior to a date and time on which the physiological fluid was taken froma subject.

In certain embodiments, for example, an increased concentration of GFAPmay be indicative of a neurological condition occurring during a time ofbetween 1 second and 5 years prior to a date and time on which thephysiological fluid was taken from a subject, for example during a timeof between 1 second and 2 years, at time of between 1 second and 1 year,a time of between 1 second and 6 months, a time of between 1 second and3 months, a time of between 1 second and 1 month, a time of between 1second and 10 days, or an increased concentration of GFAP may beindicative of a neurological condition occurring during a time ofbetween 1 second and 1 days prior to a date and time on which thephysiological fluid was taken from a subject. In certain embodiments,for example, an increased concentration of GFAP may be indicative of aneurological condition being present during a time of between 1 secondand 5 years prior to a date and time on which the physiological fluidwas taken from a subject, for example during a time of between 1 secondand 2 years, at time of between 1 second and 1 year, a time of between 1second and 6 months, a time of between 1 second and 3 months, a time ofbetween 1 second and 1 month, a time of between 1 second and 10 days, oran increased concentration of GFAP may be indicative of a neurologicalcondition being present during a time of between 1 second and 1 daysprior to a date and time on which the physiological fluid was taken froma subject.

In certain embodiments, for example, an increased concentration of UCHL1 may be indicative of a neurological condition occurring during a timeof between 1 second and 5 years prior to a date and time on which thephysiological fluid was taken from a subject, for example during a timeof between 1 second and 2 years, at time of between 1 second and 1 year,a time of between 1 second and 6 months, a time of between 1 second and3 months, a time of between 1 second and 1 month, a time of between 1second and 10 days, or an increased concentration of UCH L1 may beindicative of a neurological condition occurring during a time ofbetween 1 second and 1 days prior to a date and time on which thephysiological fluid was taken from a subject. In certain embodiments,for example, an increased concentration of UCH L1 may be indicative of aneurological condition being present during a time of between 1 secondand 5 years prior to a date and time on which the physiological fluidwas taken from a subject, for example during a time of between 1 secondand 2 years, at time of between 1 second and 1 year, a time of between 1second and 6 months, a time of between 1 second and 3 months, a time ofbetween 1 second and 1 month, a time of between 1 second and 10 days, oran increased concentration of UCH L1 may be indicative of a neurologicalcondition being present during a time of between 1 second and 1 daysprior to a date and time on which the physiological fluid was taken froma subject.

In certain embodiments, for example, an increased concentration of tauprotein may be indicative of a neurological condition occurring during atime of between 1 second and 5 years prior to a date and time on whichthe physiological fluid was taken from a subject, for example during atime of between 1 second and 2 years, at time of between 1 second and 1year, a time of between 1 second and 6 months, a time of between 1second and 3 months, a time of between 1 second and 1 month, a time ofbetween 1 second and 10 days, or an increased concentration of tauprotein may be indicative of a neurological condition occurring during atime of between 1 second and 1 days prior to a date and time on whichthe physiological fluid was taken from a subject. In certainembodiments, for example, an increased concentration of tau protein maybe indicative of a neurological condition being present during a time ofbetween 1 second and 5 years prior to a date and time on which thephysiological fluid was taken from a subject, for example during a timeof between 1 second and 2 years, at time of between 1 second and 1 year,a time of between 1 second and 6 months, a time of between 1 second and3 months, a time of between 1 second and 1 month, a time of between 1second and 10 days, or an increased concentration of tau protein may beindicative of a neurological condition being present during a time ofbetween 1 second and 1 days prior to a date and time on which thephysiological fluid was taken from a subject.

In certain embodiments, for example, an increased concentration of Abeta 40 may be indicative of a neurological condition occurring during atime of between 1 second and 5 years prior to a date and time on whichthe physiological fluid was taken from a subject, for example during atime of between 1 second and 2 years, at time of between 1 second and 1year, a time of between 1 second and 6 months, a time of between 1second and 3 months, a time of between 1 second and 1 month, a time ofbetween 1 second and 10 days, or an increased concentration of A beta 40may be indicative of a neurological condition occurring during a time ofbetween 1 second and 1 days prior to a date and time on which thephysiological fluid was taken from a subject. In certain embodiments,for example, an increased concentration of A beta 40 may be indicativeof a neurological condition being present during a time of between 1second and 5 years prior to a date and time on which the physiologicalfluid was taken from a subject, for example during a time of between 1second and 2 years, at time of between 1 second and 1 year, a time ofbetween 1 second and 6 months, a time of between 1 second and 3 months,a time of between 1 second and 1 month, a time of between 1 second and10 days, or an increased concentration of A beta 40 may be indicative ofa neurological condition being present during a time of between 1 secondand 1 days prior to a date and time on which the physiological fluid wastaken from a subject.

In certain embodiments, for example, an increased concentration of Abeta 42 may be indicative of a neurological condition occurring during atime of between 1 second and 5 years prior to a date and time on whichthe physiological fluid was taken from a subject, for example during atime of between 1 second and 2 years, at time of between 1 second and 1year, a time of between 1 second and 6 months, a time of between 1second and 3 months, a time of between 1 second and 1 month, a time ofbetween 1 second and 10 days, or an increased concentration of A beta 42may be indicative of a neurological condition occurring during a time ofbetween 1 second and 1 days prior to a date and time on which thephysiological fluid was taken from a subject. In certain embodiments,for example, an increased concentration of A beta 42 may be indicativeof a neurological condition being present during a time of between 1second and 5 years prior to a date and time on which the physiologicalfluid was taken from a subject, for example during a time of between 1second and 2 years, at time of between 1 second and 1 year, a time ofbetween 1 second and 6 months, a time of between 1 second and 3 months,a time of between 1 second and 1 month, a time of between 1 second and10 days, or an increased concentration of A beta 42 may be indicative ofa neurological condition being present during a time of between 1 secondand 1 days prior to a date and time on which the physiological fluid wastaken from a subject.

In certain embodiments, for example, an increased concentration of S100Bmay be indicative of a neurological condition occurring during a time ofbetween 1 second and 5 years prior to a date and time on which thephysiological fluid was taken from a subject, for example during a timeof between 1 second and 2 years, at time of between 1 second and 1 year,a time of between 1 second and 6 months, a time of between 1 second and3 months, a time of between 1 second and 1 month, a time of between 1second and 10 days, or an increased concentration of S100B may beindicative of a neurological condition occurring during a time ofbetween 1 second and 1 days prior to a date and time on which thephysiological fluid was taken from a subject. In certain embodiments,for example, an increased concentration of S100B may be indicative of aneurological condition being present during a time of between 1 secondand 5 years prior to a date and time on which the physiological fluidwas taken from a subject, for example during a time of between 1 secondand 2 years, at time of between 1 second and 1 year, a time of between 1second and 6 months, a time of between 1 second and 3 months, a time ofbetween 1 second and 1 month, a time of between 1 second and 10 days, oran increased concentration of S100B may be indicative of a neurologicalcondition being present during a time of between 1 second and 1 daysprior to a date and time on which the physiological fluid was taken froma subject.

In certain embodiments, for example, an increased concentration of NSEmay be indicative of a neurological condition occurring during a time ofbetween 1 second and 5 years prior to a date and time on which thephysiological fluid was taken from a subject, for example during a timeof between 1 second and 2 years, at time of between 1 second and 1 year,a time of between 1 second and 6 months, a time of between 1 second and3 months, a time of between 1 second and 1 month, a time of between 1second and 10 days, or an increased concentration of NSE may beindicative of a neurological condition occurring during a time ofbetween 1 second and 1 days prior to a date and time on which thephysiological fluid was taken from a subject. In certain embodiments,for example, an increased concentration of NSE may be indicative of aneurological condition being present during a time of between 1 secondand 5 years prior to a date and time on which the physiological fluidwas taken from a subject, for example during a time of between 1 secondand 2 years, at time of between 1 second and 1 year, a time of between 1second and 6 months, a time of between 1 second and 3 months, a time ofbetween 1 second and 1 month, a time of between 1 second and 10 days, oran increased concentration of NSE may be indicative of a neurologicalcondition being present during a time of between 1 second and 1 daysprior to a date and time on which the physiological fluid was taken froma subject.

In certain embodiments, for example, the at least one parameter maycomprise a ratio of a concentration of a first biomarker to aconcentration of a second component (for example a non-CNS protein or asecond biomarker (for example a second biomarker comprising a CNSprotein)) of the fluid sample. In certain embodiments, for example, theratio may be indicative of the neurological condition at a value of atleast 1% (for example at least 2%, at least 4%, at least 6%, at least8%, at least 10%, at least 12%, at least 14%, at least 15%, at least20%, at least 25%, at least 30%, at least 35%, at least 40%, at least45%), at least 50%, at least 55%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 99%, at least 100%, at least 110%, at least 115%, at least120%, at least 140%, at least 150%, at least 200%, at least 250%, atleast 300%, at least 350%, at least 400%, or between 1% and 400%,between 1% and 300%, between 1% and 200%, between 1% and 100%, between1% and 50%, between 1% and 25%, between 1% and 10%, between 10% and400%, between 10% and 300%, between 10% and 200%, between 10% and 100%,between 10% and 50%, between 50% and 400%, between 50% and 300%, between50% and 200%, between 50% and 100%, between 50% and 75%, between 75% and100%, or 1%, 2%, 4%, 5%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%,105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%,165%, 170%, 175%, 180%, 185%, 190%, 195%, 200%, 250%, 300%, 350%, or400%. In certain embodiments, for example, the ratio may be indicativeof the neurological condition at a value of between 1% and 500%, between1% and 450%, between 1% and 400%, between 1% and 350%, between 1% and300%, between 1% and 250%, between 1% and 200%, between 1% and 150%,between 1% and 100%, between 1% and 50%, between 1% and 25%, between 1%and 20%, between 1% and 15%, between 1% and 10%, between 1% and 5%,between 2% and 500%, between 2% and 450%, between 2% and 400%, between2% and 350%, between 2% and 300%, between 2% and 250%, between 2% and200%, between 2% and 150%, between 2% and 100%, between 2% and 50%,between 2% and 25%, between 2% and 20%, between 2% and 15%, between 2%and 10%, between 5% and 500%, between 5% and 450%, between 5% and 400%,between 5% and 350%, between 5% and 300%, between 5% and 250%, between5% and 200%, between 5% and 150%, between 5% and 100%, between 5% and50%, between 5% and 25%, between 5% and 20%, between 5% and 15%, between5% and 10%, between 10% and 500%, between 10% and 450%, between 10% and400%, between 10% and 350%, between 10% and 300%, between 10% and 250%,between 10% and 200%, between 10% and 150%, between 10% and 100%,between 10% and 50%, between 10% and 25%, between 10% and 20%, between10% and 15%, between 15% and 500%, between 15% and 450%, between 15% and400%, between 15% and 350%, between 15% and 300%, between 15% and 250%,between 15% and 200%, between 15% and 150%, between 15% and 100%,between 15% and 50%, between 15% and 25%, between 15% and 20%, between20% and 500%, between 20% and 450%, between 20% and 400%, between 20%and 350%, between 20% and 300%, between 20% and 250%, between 20% and200%, between 20% and 150%, between 20% and 100%, between 20% and 50%,between 20% and 25%, between 25% and 500%, between 25% and 450% between25% and 400%, between 25% and 350%, between 25% and 300%, between 25%and 250%, between 25% and 200%, between 25% and 150%, between 25% and100%, between 25% and 50%, between 50% and 500%, between 50% and 450%,between 50% and 400%, between 50% and 350%, between 50% and 300%,between 50% and 250%, between 50% and 200%, between 50% and 150%,between 50% and 100%, between 100% and 500%, between 100% and 450%,between 100% and 400%, between 100% and 350%, between 100% and 300%,between 100% and 250%, between 100% and 200%, between 100% and 150%,between 150% and 500%, between 150% and 450%, between 150% and 400%,between 150% and 350%, between 150% and 300%, between 150% and 250%,between 150% and 200%, between 200% and 500%, between 200% and 450%,between 200% and 400%, between 200% and 350%, between 200% and 300%,between 200% and 250%, between 250% and 500%, between 250% and 450%,between 250% and 400%, between 250% and 350%, between 250% and 300%,between 300% and 500%, between 300% and 450%, between 300% and 400%,between 300% and 350%, between 350% and 500%, between 350% and 450%,between 350% and 400%, between 400% and 500%, between 400% and 450%, orbetween 450% and 500%.

In certain embodiments, for example, the at least one parameter maycomprise an increase (for example a percentage increase) in theconcentration of a biomarker, for example a percentage increase of atleast 1% (for example at least 2%, at least 4%, at least 6%, at least8%, at least 10%, at least 12%, at least 14%, at least 15%, at least20%, at least 25%, at least 30%, at least 35%, at least 40%, at least45%), at least 50%, at least 55%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 99%, at least 100%, at least 110%, at least 115%, at least120%, at least 140%, at least 150%, at least 200%, at least 250%, atleast 300%, at least 350%, at least 400%, or between 1% and 400%,between 1% and 300%, between 1% and 200%, between 1% and 100%, between1% and 50%, between 1% and 25%, between 1% and 10%, between 10% and400%, between 10% and 300%, between 10% and 200%, between 10% and 100%,between 10% and 50%, between 50% and 400%, between 50% and 300%, between50% and 200%, between 50% and 100%, between 50% and 75%, between 75% and100%, or 1%, 2%, 4%, 5%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%,105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%,165%, 170%, 175%, 180%, 185%, 190%, 195%, 200%, 250%, 300%, 350%, or400%. In certain embodiments, for example, the at least one parametermay comprise a percentage increase in the concentration of a biomarkerof between 1% and 500%, between 1% and 450%, between 1% and 400%,between 1% and 350%, between 1% and 300%, between 1% and 250%, between1% and 200%, between 1% and 150%, between 1% and 100%, between 1% and50%, between 1% and 25%, between 1% and 20%, between 1% and 15%, between1% and 10%, between 1% and 5%, between 2% and 500%, between 2% and 450%,between 2% and 400%, between 2% and 350%, between 2% and 300%, between2% and 250%, between 2% and 200%, between 2% and 150%, between 2% and100%, between 2% and 50%, between 2% and 25%, between 2% and 20%,between 2% and 15%, between 2% and 10%, between 5% and 500%, between 5%and 450%, between 5% and 400%, between 5% and 350%, between 5% and 300%,between 5% and 250%, between 5% and 200%, between 5% and 150%, between5% and 100%, between 5% and 50%, between 5% and 25%, between 5% and 20%,between 5% and 15%, between 5% and 10%, between 10% and 500%, between10% and 450%, between 10% and 400%, between 10% and 350%, between 10%and 300%, between 10% and 250%, between 10% and 200%, between 10% and150%, between 10% and 100%, between 10% and 50%, between 10% and 25%,between 10% and 20%, between 10% and 15%, between 15% and 500%, between15% and 450%, between 15% and 400%, between 15% and 350%, between 15%and 300%, between 15% and 250%, between 15% and 200%, between 15% and150%, between 15% and 100%, between 15% and 50%, between 15% and 25%,between 15% and 20%, between 20% and 500%, between 20% and 450%, between20% and 400%, between 20% and 350%, between 20% and 300%, between 20%and 250%, between 20% and 200%, between 20% and 150%, between 20% and100%, between 20% and 50%, between 20% and 25%, between 25% and 500%,between 25% and 450%, between 25% and 400%, between 25% and 350%,between 25% and 300%, between 25% and 250%, between 25% and 200%,between 25% and 150%, between 25% and 100%, between 25% and 50%, between50% and 500%, between 50% and 450%, between 50% and 400%, between 50%and 350%, between 50% and 300%, between 50% and 250%, between 50% and200%, between 50% and 150%, between 50% and 100%, between 100% and 500%,between 100% and 450%, between 100% and 400%, between 100% and 350%,between 100% and 300%, between 100% and 250%, between 100% and 200%,between 100% and 150%, between 150% and 500%, between 150% and 450%,between 150% and 400%, between 150% and 350%, between 150% and 300%,between 150% and 250%, between 150% and 200%, between 200% and 500%,between 200% and 450%, between 200% and 400%, between 200% and 350%,between 200% and 300%, between 200% and 250%, between 250% and 500%,between 250% and 450%, between 250% and 400%, between 250% and 350%,between 250% and 300%, between 300% and 500%, between 300% and 450%,between 300% and 400%, between 300% and 350%, between 350% and 500%,between 350% and 450%, between 350% and 400%, between 400% and 500%,between 400% and 450%, or between 450% and 500%, for example as comparedto a reference level.

In certain embodiments, for example, the at least one parameter maycomprise a reduction (for example a percentage reduction) in theconcentration of a biomarker of between 1% and 99%, for example between1% and 95%, between 1% and 90%, between 1% and 85%, between 1% and 80%,between 1% and 75%, between 1% and 70%, between 1% and 65%, between 1%and 60%, between 1% and 55%, between 1% and 50%, between 1% and 45%,between 1% and 40%, between 1% and 35%, between 1% and 30%, between 1%and 25%, between 1% and 20%, between 1% and 15%, between 1% and 10%,between 1% and 5%, between 5% and 99%, between 5% and 95%, between 5%and 90%, between 5% and 85%, between 5% and 80%, between 5% and 75%,between 5% and 70%, between 5% and 65%, between 5% and 60%, between 5%and 55%, between 5% and 50%, between 5% and 45%, between 5% and 40%,between 5% and 35%, between 5% and 30%, between 5% and 25%, between 5%and 20%, between 5% and 15%, between 5% and 10%, between 10% and 99%,between 10% and 95%, between 10% and 90%, between 10% and 85%, between10% and 80%, between 10% and 75%, between 10% and 70%, between 10% and65%, between 10% and 60%, between 10% and 55%, between 10% and 50%,between 10% and 45%, between 10% and 40%, between 10% and 35%, between10% and 30%, between 10% and 25%, between 10% and 20%, between 10% and15%, between 15% and 99%, between 15% and 95%, between 15% and 90%,between 15% and 85%, between 15% and 80%, between 15% and 75%, between15% and 70%, between 15% and 65%, between 15% and 60%, between 15% and55%, between 15% and 50%, between 15% and 45%, between 15% and 40%,between 15% and 35%, between 15% and 30%, between 15% and 25%, between15% and 20%, between 20% and 99%, between 20% and 95%, between 20% and90%, between 20% and 85%, between 20% and 80%, between 20% and 75%,between 20% and 70%, between 20% and 65%, between 20% and 60%, between20% and 55%, between 20% and 50%, between 20% and 45%, between 20% and40%, between 20% and 35%, between 20%) and 30%, between 20% and 25%,between 25% and 99%, between 25% and 95%, between 25% and 90%, between25% and 85%, between 25% and 80%, between 25% and 75%, between 25% and70%, between 25% and 65%, between 25% and 60%, between 25% and 55%,between 25% and 50%, between 25% and 45%, between 25% and 40%, between25% and 35%, between 25% and 30%, between 30% and 99%, between 30% and95%, between 30% and 90%, between 30% and 85%, between 30% and 80%,between 30% and 75%, between 30% and 70%, between 30% and 65%, between30% and 60%, between 30% and 55%, between 30% and 50%, between 30% and45%, between 30% and 40%, between 30% and 35%, between 35% and 99%,between 35% and 95%, between 35% and 90%, between 35% and 85%, between35% and 80%, between 35% and 75%, between 35% and 70%, between 35% and65%, between 35% and 60%, between 35% and 55%, between 35% and 50%,between 35% and 45%, between 35% and 40%, between 40% and 99%, between40% and 95%, between 40% and 90%, between 40% and 85%, between 40% and80%, between 40% and 75%, between 40% and 70%, between 40% and 65%,between 40% and 60%, between 40% and 55%, between 40% and 50%, between40% and 45%, between 45% and 99%, between 45% and 95%, between 45% and90%, between 45% and 85%, between 45% and 80%, between 45% and 75%,between 45% and 70%, between 45% and 65%, between 45% and 60%, between45% and 55%, between 45% and 50%, between 50% and 99%, between 50% and95%, between 50% and 90%, between 50% and 85%, between 50% and 80%,between 50% and 75%, between 50% and 70%, between 50% and 65%, between50% and 60%, between 50% and 55%, between 55% and 99%, between 55% and95%, between 55% and 90%, between 55% and 85%, between 55% and 80%,between 55% and 75%, between 55% and 70%, between 55% and 65%, between55% and 60%, between 60% and 99%, between 60% and 95%, between 60% and90%, between 60% and 85%, between 60% and 80%, between 60% and 75%,between 60% and 70%, between 60% and 65%, between 65% and 99%, between65% and 95%, between 65% and 90%, between 65% and 85%, between 65% and80%, between 65% and 75%, between 65% and 70%, between 70% and 99%,between 70% and 95%, between 70% and 90%, between 70% and 85%, between70% and 80%, between 70% and 75%, between 75% and 99%, between 75% and95%, between 75% and 90%, between 75% and 85%, between 75% and 80%,between 80% and 99%, between 80% and 95%, between 80% and 90%, between80% and 85%, between 85% and 99%, between 85% and 95%, between 85% and90%, between 90% and 99%, between 90% and 95%, or between 95% and 99%.

A schematic depiction of a method to quantify concentrations of fouranalytes (for example NF-L, GFAP, UCH L1, and Tau) in a liquid samplevia digital assay is shown in FIG. 1. The liquid sample 100 (in vessel102) is provided 104 containing first type of analyte molecule (forexample a first analyte molecule 106 such as NF-L), a second type ofanalyte molecule (for example a second analyte molecule 108 such asGFAP), a third type of analyte molecule (for example a third analytemolecule 110 such as UCH L1), and a fourth type of analyte molecule (forexample a fourth analyte molecule 112 such as Tau). For example, thefirst type of analyte molecule may be NF-L, the second type of analytemolecule may be GFAP, the third type of analyte molecule may be UCH L1,and the fourth type of analyte molecule may be Tau. Capture objectsincluding a first capture object 114 having a first anti-analyteimmobilization agent 116, a second capture object 118 having a secondanti-analyte immobilization agent 120, a third capture object 122 havinga third anti-analyte immobilization agent 124, and a fourth captureobject 126 having a fourth anti-analyte immobilization agent 128 arecombined 130 with the liquid sample 100, wherein the first anti-analyteimmobilization agent 116 is specific to the first type of analytemolecule, the second anti-analyte immobilization agent 120 is specificto the second type of analyte molecule, the third anti-analyteimmobilization agent 124 is specific to the third type of analytemolecule, and the fourth anti-analyte immobilization agent 128 isspecific to the fourth type of analyte molecule. The added captureobjects are incubated 132 with the liquid sample 100 for a period oftime, resulting in the first capture object 114 binding with the firstanalyte molecule 106, the second capture object 118 binding with thesecond analyte molecule 108, the third capture object 122 binding withthe third analyte molecule 110, and the fourth capture object 126binding with the fourth analyte molecule 112. Some of the captureobjects (not shown) do not bind with any analyte. An excess number ofcapture objects (not shown) is provided whereby the fraction of captureobjects binding with more than one analyte is statisticallyinsignificant. After the capture objects have immobilized at least aportion of the analyte molecules (not all analyte molecules are shown),a first type of detectable agent (for example a first detectable agent134) configured to bind with the first type of analyte, a second type ofdetectable agent (for example a second detectable agent 136) configuredto bind with the second type of analyte, a third type of detectableagent (for example a third detectable agent 138) configured to bind withthe third type of analyte, and a fourth type of detectable agent (forexample a fourth detectable agent 140) configured to bind with thefourth type of analyte are contacted 142 with the capture objects andincubated 144, resulting in the first detectable agent 134 binding tothe first analyte molecule 106, the second detectable agent 136 bindingto the second analyte molecule 108, the third detectable agent 138binding to the third analyte molecule 110, and the fourth detectableagent 140 binding to the fourth analyte molecule 112. Detectable agentsdo not bind to a capture object unless the capture object hasimmobilized an analyte molecule (not all detectable agents are shown).The capture objects are spatially separated 146 into a plurality ofreaction vessels (for example femtoliter-sized reaction vessels etchedin a plate) on a substrate 148. As shown, the first capture object 114,the second capture object 118, the third capture object 122, and thefourth capture object 126 (along with additional capture objects notshown) are present in separate reaction vessels while some of thereaction vessels such as the reaction vessel 150 may not contain anycapture objects. The substrate 148 may then be analyzed 152 by anoptical-based analyzer 154 to determine the number of reaction vesselscontaining an analyte bound to a capture object, wherein in the numberof the first type of analyte molecule, the number of the second type ofanalyte molecule, the number of the third type of analyte molecule, andthe number of the fourth type of analyte molecule may be related to ameasures of the concentration of each type of analyte in the liquidsample 100 (for example by a standard curve).

A schematic depiction of a method to quantify concentrations of fouranalytes (for example NF-L, GFAP, UCH L1, and Tau) in a liquid samplevia a multi-spotted reaction well (for example one well of a pluralityof such wells) is shown in FIG. 2. The liquid sample 100 (in vessel 102)is provided 100 containing first type of analyte molecule (for example afirst analyte molecule 106), a second type of analyte molecule (forexample a second analyte molecule 108), a third type of analyte molecule(for example a third analyte molecule 110), and a fourth type of analytemolecule (for example a fourth analyte molecule 112). For example, thefirst type of analyte molecule may be NF-L, the second type of analytemolecule may be GFAP, the third type of analyte molecule may be UCH L1,and the fourth type of analyte molecule may be Tau. Anti-analyteimmobilization agents including a first type of anti-analyteimmobilization agent (for example the anti-analyte immobilization agent200), a second type of anti-analyte immobilization agent (for examplethe anti-analyte immobilization agent 202), a third type of anti-analyteimmobilization agent (for example the anti-analyte immobilization agent204), and a fourth type of anti-analyte immobilization agent (forexample the anti-analyte immobilization agent 206), that are immobilizedin a plurality of spatially separated zones (or “spots”) in a reactionwell 208. For example a first spot 210 contains a plurality of the firsttype of anti-analyte immobilization agent exclusive of the other typesof anti-analyte immobilization agents; a second spot 212 contains aplurality of the second type of anti-analyte immobilization agentexclusive of the other types of anti-analyte immobilization agents; athird spot 214 contains a plurality of the third type of anti-analyteimmobilization agent exclusive of the other types of anti-analyteimmobilization agents; and a fourth spot 216 contains a plurality of thefourth type of anti-analyte immobilization agent exclusive of the othertypes of anti-analyte immobilization agents. The liquid sample 100 isadded 218 to the reaction well 208, wherein the first anti-analyteimmobilization agent 200 is specific to the first type of analytemolecule, the second anti-analyte immobilization agent 202 is specificto the second type of analyte molecule, the third anti-analyteimmobilization agent 204 is specific to the third type of analytemolecule, and the fourth anti-analyte immobilization agent 206 isspecific to the fourth type of analyte molecule. The anti-analyteimmobilization agents are incubated 220 with the liquid sample 100 for aperiod of time, resulting in the first anti-analyte immobilization agent200 binding with the first analyte molecule 106, the second anti-analyteimmobilization agent 202 binding with the second analyte molecule 108,the third anti-analyte immobilization agent 204 binding with the thirdanalyte molecule 110, and the fourth anti-analyte immobilization agent206 binding with the fourth analyte molecule 112. In certainembodiments, for example, the reaction well may be agitated according toa predetermined scheme to increase fluid flow of the liquid samplerelative to the one or more spatially separated zones and therebyachieve one or more of increased sensitivity (for example lower LOQ),reduced assay time, and assay reproducibility. After the anti-analyteimmobilization agents have immobilized at least a portion of the analytemolecules (not all analyte molecules are shown), a first type ofdetectable agent (for example a first detectable agent 222) configuredto bind with the first type of analyte, a second type of detectableagent (for example a second detectable agent 224) configured to bindwith the second type of analyte, a third type of detectable agent (forexample a third detectable agent 226) configured to bind with the thirdtype of analyte, and a fourth type of detectable agent (for example afourth detectable agent 228) configured to bind with the fourth type ofanalyte are contacted 230 with the plurality of spatially separatedzones and incubated 232 in the reaction well 208, resulting in the firstdetectable agent 222 binding to the first analyte molecule 106, thesecond detectable agent 224 binding to the second analyte molecule 108,the third detectable agent 226 binding to the third analyte molecule110, and the fourth detectable agent 228 binding to the fourth analytemolecule 112. Detectable agents do not bind to an anti-analyteimmobilization agent unless the anti-analyte immobilization agent hasimmobilized an analyte molecule (not all detectable agents molecules areshown). The substrate 234 may then be analyzed 236 by an optical-basedanalyzer 238 to determine the signal levels in spatially separatedreaction vessels containing an analyte bound to an anti-analyteimmobilization agent, wherein in the signal level of the first type ofanalyte molecule, the signal level of the second type of analytemolecule, the signal level of the third type of analyte molecule, andthe signal level of the fourth type of analyte molecule may be relatedto a measures of the concentration of each type of analyte in the liquidsample 100. For example, the signal may comprise a chemilluminecentsignal. In certain embodiments, for example, analyte quantification viathe described detection of analytes in the spotted reaction well mayprovide one or more of the same level of sensitivity, LOQ, and LOD asone or more of the digital assays described herein or in one of theINCORPORATED REFERENCES.

Any of the disclosed methods, tests, assays, kits, or systems maycomprising processing the results of one or more assays (for example theconcentrations of one or more biomarkers) into useful informationregarding a risk, diagnosis, prognosis, or state of a neurologicalcondition in a subject (for example a human subject such as a neonate)by passing the results through a classification model to compute atleast one classification value which can be compared to at least onethreshold value.

In certain embodiments, for example, the at least one threshold valuemay correspond to a predetermined sensitivity level for a multivariatestatistical model of the expression levels of one or more biomarkers ina plurality (for example 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10) oftraining samples from individuals diagnosed as having an indicatedneurological condition (for example TBI) and control samples fromindividuals without the indicated neurological condition. In certainembodiments, for example, the subject's classification value may becalculated using the multivariate statistical model. In certainembodiments, for example, the multivariate statistical model maycomprise a binary logistic regression, a linear regression, a quadraticregression, a polynomial regression, a logistic regression, results of aprincipal component analysis, results of a maximum likelihood analysis,a neural network, results of a linear discriminant analysis, a decisiontree, or a combination of two or more of the foregoing.

Certain embodiments, for example, may provide methods, tests, assays,kits, or systems to measure NF-L, GFAP, UCH L1, and Tau. In certainembodiments, for example, NF-L, GFAP, UCH L1, and Tau may be detected bya multiplex assay. In certain embodiments, for example, the multiplexassay may comprise adding portions of a liquid sample (for example aliquid sample obtained by diluting a sample of human physiological fluidsuch as human blood serum or plasma) to a plurality of reaction wells(for example a 96-well assay plate). In certain embodiments, forexample, each of the reaction wells may comprise a plurality of zones(or spots). In certain embodiments, for example, the plurality of zones(or spots) may be spatially separated. In certain embodiments, forexample, the zones by be radially disposed (for example symmetricallydisposed at a fixed radius about a central position in each reactionwell). In certain embodiments, for example, each of the reaction wellsmay be sized to contain, in total, between 20 mcL and 100 mcL (forexample 50 mcL) of liquid sample and assay reagents. In certainembodiments, for example, each of the zones may be coated (or covalentlyconjugated to) a plurality (for example 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore than 10) of an anti-analyte agent that is specific to one of NF-L,GFAP, UCH L1, and Tau, whereby a first zone of each reaction well willpreferentially immobilize NF-L, a second zone of each reaction well willpreferentially immobilize GFAP, a third zone of each reaction well willpreferentially immobilize UCH L1, and a fourth zone of each reactionwell will preferentially immobilize Tau. In certain embodiments, forexample, the multi-well plate may be coupled to a digital nanofluidicdeposition system for controlled addition of sample, washing reagents,and detection agents. In certain embodiments, for example, detectableagents (for example four different detectable agents having differentcolors or the same color) may be added to each of the reaction wells tobind with immobilized NF-L, GFAP, UCH L1, and Tau and produce adetectable signal. In certain embodiments, for example, the multi-wellplate may made of translucent plastic. In certain embodiments, forexample, imaging of detectable signals may be performed through thebottom of the translucent multi-well plate. In certain embodiments, forexample, the multi-well plate may be coupled to an agitation system. Incertain embodiments, for example, the agitation system may be configuredto agitate the multi-well plate to form a vortex of fluid in eachreaction well.

In certain embodiments, for example, the multiplex assay may compriseagitating the liquid sample, for example agitating the liquid sample forup to 1 hour, up to 1.5 hours, up to 2 hours, up to 2.5 hours, up to 3hours, up to 3.5 hours, up to 4 hours, up to 4.5 hours, up to 5 hours,up to 6 hours, up to 8 hours, up to 10 hours, up to between 1 hour and 3hours, up to between 1.5 hours and 2.5 hours, for 2 hours, or forgreater than 10 hours. In certain embodiments, for example the multiplexassay may comprise adding biotinylated detection antibodies, followed byan additional 30 minutes of shaking. In certain embodiments, forexample, the detection antibodies may bind to form an amino complex. Incertain embodiments, for example, horseradish peroxidase(HRP)-conjugated streptavidin may be added, followed by an additional 30minute agitation cycle, substrate added, and the zones containing NF-L,GFAP, UCH L1, and Tau detected via chemiluminescent signal. In certainembodiments, for example, a machine learning algorithm may optimizeexposure times in the number of images for each sample to maximizesensitivity and dynamic range. In certain embodiments, for example, themultiplex assay may be performed within 6 hours inclusive of preparationof a the liquid sample from a sample of physiological fluid, for examplewithin 5 hours, within 4 hours, within 3 hours within 2 hours, within 1hour, within 30 minutes, within between 1 hour and 6 hours, withinbetween 2 hours and 4 hours, within between 2.5 hours and 3.5 hours, orwithin 3 hours.

Certain embodiments may provide, for example, methods, tests, assays,kits, or systems for determining a measure of the concentration of apanel of neurological biomarkers in a sample derived from a human aredisclosed. In certain embodiments, for example, the methods, tests,assays, kits, or systems may involve determining the concentration oftwo or more biomarkers associated with brain injury and/orneurodegeneration in a human sample. In certain embodiments, forexample, the method may comprise determining a measure of theconcentration of NF-L and at least one other biomarker selected from thegroup consisting of GFAP, UCH L1, and Tau. An exemplary method and kitmay be directed to determination of a measure of the concentration of abiomarker panel comprising at least NF-L, GFAP, UCH L1, and Tau. Incertain embodiments, for example, a measure of the concentration of thebiomarkers may be determined using a single assay. In some such cases,the assay may utilize certain assay conditions that allow for thedetermination of each biomarker with a relatively high specificity andsensitivity. The methods, tests, assays, kits, and systems describedherein may be used to assess a variety of brain injuries andneurodegenerative conditions, including TBI.

A cascade of biomarkers, such as NF-L, GFAP, UCH L1, and Taus, may begenerated in the brain in response to and/or in proportion to the extentof a brain injury. NF-L, a cytoskeletal intermediate filament protein,may combine with other proteins to form neurofilaments in neurons andmay be released in significant quantity following axonal damage orneuronal degeneration. Tau protein, a microtubule-stabilizing proteinprimarily localized in CNS neurons, may be observed in the cerebrospinalfluid (CSF) of patients with neurodegenerative disease and headinjuries, suggesting its extracellular release during neuronal damageand a role as a biomarker with specificity for brain injury. GFAP, aclass-III intermediate filament involved in many central nervous systemprocesses including cell communication and the functioning of the bloodbrain barrier, may be associated with multiple diseases such as TBI,stroke, brain tumors, etc. UCH L1, which hydrolyzes small C-terminaladducts of ubiquitin to generate the ubiquitin monomer and is expressedpredominantly in neurons, may be released from injured neurons and flowinto the cerebrospinal fluid and circulating blood. Such biomarkerscould in turn diffuse across the blood brain barrier and into the bloodin response to and/or in proportion to the extent of the injury, and maybe generally found in low abundance. In certain embodiments, forexample, the ability to determine a measure of the concentration of twoor more biomarkers (for example three or more biomarkers, four or morebiomarkers) in a patient sample (or a plurality samples) obtainedfollowing a suspected injury event may be used to determine whetherbrain injury occurred and/or otherwise assess the injury. For example, ameasure of the concentration of two or more biomarkers may be used toassess the severity of the brain injury.

In certain embodiments, for example, sample(s) of the patient'scerebrospinal fluid (CSF) may be obtained and analyzed to determine ameasure of the concentration of the biomarkers. In certain embodiments,for example, it may be advantageous to determine the level of biomarkersin the blood of a patient as compared to CSF, as blood sampling may begenerally less invasive and may result in fewer complications ascompared to CSF sampling. However, many of the biomarkers that arepresent in the CSF have a slow rate of transmission across and/or a highbarrier of transportation across the blood-brain barrier (BBB) and thus,a measure of the concentration of the biomarkers in the patient's bloodmay be generally sufficiently lower as compared to a measure of theconcentration in CSF, which can make it difficult or impossible toaccurately determine using typically employed conventional immunoassays.Certain embodiments may provide, for example, methods, tests, assays,kits, or systems that have very low limits of quantification (LOQ)and/or limits of detection (LOD) can facilitate determination of ameasure of the concentration of such biomarkers in the patient's bloodwith sufficient accuracy and repeatability to provide statisticallysignificant and/or meaningful results. In certain embodiments, forexample, the methods, tests, assays, kits, or systems may have very lowLODs and/or LOQs (for example in the low pg/mL range) to determine ameasure of the concentration of two or more biomarkers in a sampleobtained from a patient following a suspected injury event. In certainembodiments, for example, one or more parameters related to theconcentrations of the biomarkers in the sample (for example bloodsample) may be correlated with the diagnosis of brain injury, theassessment of the extent of brain injury, and/or a method of treatmentfollowing the injury event.

It should be noted, that while many of the embodiments described hereinfocus on brain injuries caused by traumatic events, this is by no waylimiting, and In certain embodiments, for example, the brain injury maybe caused by other events, for example, a biochemical event, such asoxygen deprivation (hypoxia). Hypoxia generally refers to a deficiencyin the amount of oxygen reaching body tissues or a condition ofinsufficient levels of oxygen in issue or blood. Oxygen deprivation tothe brain results in neuronal damage and death, which may be in turnrelated to the extent of long term brain dysfunction.

In certain embodiments, for example, the methods, tests, assays, kits,or systems may comprise determining a measure of the concentration oftwo or more biomarkers selected from the group consisting of GFAP, UCHL1, Tau, and NF-L. In certain embodiments, for example, the methods,tests, assays, kits, or systems may comprise determining a measure ofthe concentration of NF-L protein and at least one other biomarkerselected from the group consisting of GFAP, UCH L1, and Tau. In certainembodiments, for example, the methods, tests, assays, kits, or systemsmay comprise determining a measure of the concentration of NF-L andGFAP. In certain embodiments, for example, the method may comprisedetermining a measure of the concentration of NF-L and UCH L1. Incertain embodiments, for example, the methods, tests, assays, kits, orsystems may comprise determining a measure of the concentration of NF-Land Tau protein. In certain embodiments, for example, the methods,tests, assays, kits, or systems may comprise determining a measure ofthe concentration of NF-L and A beta 40. In certain embodiments, forexample, the methods, tests, assays, kits, or systems may comprisedetermining a measure of the concentration of NF-L and A beta 42. Incertain embodiments, for example, the methods, tests, assays, kits, orsystems may comprise determining a measure of the concentration of NF-Land S100B. In certain embodiments, for example, the methods, tests,assays, kits, or systems may comprise determining a measure of theconcentration of NF-L and NSE.

In certain embodiments, for example, the methods, tests, assays, kits,or systems may comprise determining a measure of the concentration ofGFAP and at least one other biomarker selected from the group consistingof UCH L1, Tau, A beta 40, A beta 42, S100B, and NSE. In certainembodiments, for example, the methods, tests, assays, kits, or systemsmay comprise determining a measure of the concentration of GFAP and UCHL1. In certain embodiments, for example, the methods, tests, assays,kits, or systems may comprise determining a measure of the concentrationof GFAP and Tau. In certain embodiments, for example, the methods,tests, assays, kits, or systems may comprise determining a measure ofthe concentration of GFAP and A beta 40. In certain embodiments, forexample, the methods, tests, assays, kits, or systems may comprisedetermining a measure of the concentration of GFAP and A beta 42. Incertain embodiments, for example, the methods, tests, assays, kits, orsystems may comprise determining a measure of the concentration of GFAPand S100B. In certain embodiments, for example, the methods, tests,assays, kits, or systems may comprise determining a measure of theconcentration of GFAP and NSE.

In certain embodiments, for example, the methods, tests, assays, kits,or systems may comprise determining a measure of the concentration ofUCH L1 and at least one other biomarker selected from the groupconsisting of Tau, A beta 40, A beta 42, S100B, and NSE. In certainembodiments, for example, the methods, tests, assays, kits, or systemsmay comprise determining a measure of the concentration of UCH L1 andTau. In certain embodiments, for example, the methods, tests, assays,kits, or systems may comprise determining a measure of the concentrationof UCH L1 and A beta 40. In certain embodiments, for example, themethods, tests, assays, kits, or systems may comprise determining ameasure of the concentration of UCH L1 and A beta 42. In certainembodiments, for example, the methods, tests, assays, kits, or systemsmay comprise determining a measure of the concentration of UCH L1 andS100B. In certain embodiments, for example, the methods, tests, assays,kits, or systems may comprise determining a measure of the concentrationof UCH L1 and NSE.

In certain embodiments, for example, the methods, tests, assays, kits,or systems may comprise determining a measure of the concentration ofTau and at least one other biomarker selected from the group consistingof A beta 40, A beta 42, S100B, and NSE. In certain embodiments, forexample, the methods, tests, assays, kits, or systems may comprisedetermining a measure of the concentration of Tau and A beta 40. Incertain embodiments, for example, the methods, tests, assays, kits, orsystems may comprise determining a measure of the concentration of Tauand A beta 42. In certain embodiments, for example, the methods, tests,assays, kits, or systems may comprise determining a measure of theconcentration of Tau and S100B. In certain embodiments, for example, themethods, tests, assays, kits, or systems may comprise determining ameasure of the concentration of Tau and NSE.

In certain embodiments, for example, the methods, tests, assays, kits,or systems may comprise determining a measure of the concentration of Abeta 40 and at least one other biomarker selected from the groupconsisting of A beta 42, S100B, and NSE. In certain embodiments, forexample, the methods, tests, assays, kits, or systems may comprisedetermining a measure of the concentration of A beta 40 and A beta 42.In certain embodiments, for example, the methods, tests, assays, kits,or systems may comprise determining a measure of the concentration of Abeta 40 and S100B. In certain embodiments, for example, the methods,tests, assays, kits, or systems may comprise determining a measure ofthe concentration of A beta 40 and NSE.

In certain embodiments, for example, the methods, tests, assays, kits,or systems may comprise determining a measure of the concentration of Abeta 42 and at least one other biomarker selected from the groupconsisting of S100B, and NSE. In certain embodiments, for example, themethods, tests, assays, kits, or systems may comprise determining ameasure of the concentration of A beta 42 and S100B. In certainembodiments, for example, the methods, tests, assays, kits, or systemsmay comprise determining a measure of the concentration of A beta 42 andNSE.

In certain embodiments, for example, the methods, tests, assays, kits,or systems may comprise determining a measure of the concentration ofS100B and NSE.

In certain embodiments, for example, the methods, tests, assays, kits,or systems may comprise measuring a selected number and combination ofbiomarkers associated with brain injury and/or neurodegeneration. Incertain embodiments, for example, the methods, tests, assays, kits, orsystems may comprise determining the concentration of two biomarkers. Incertain embodiments, for example, the methods, tests, assays, kits, orsystems may comprise determining the concentration of three biomarkers.In certain embodiments, for example, the methods, tests, assays, kits,or systems may comprise determining a measure of the concentration of atleast the following four biomarkers: Tau, GFAP, UCH L1, and NF-L. Incertain embodiments, for example, the methods, tests, assays, kits, orsystems may comprise determining the concentration of at least about 1biomarker, at least about 2, at least about 3, at least about 4, atleast about 5, at least about 6, at least about 7, at least about 8, atleast about 9, or at least about 10 biomarkers. In certain embodiments,for example, the number of biomarkers, whose concentration may bedetermined, may range from 2 to 10, from 2 to 8, ranges from 2 to 6, orfrom 2 to 5 (for example from 2 to 4).

In certain embodiments, for example, an immunoassay may be used todetermine a measure of the concentration of two or more biomarkers. Incertain embodiments, for example, the immunoassay may be a multiplexassay in which the concentration of more than one biomarker may bemeasured in a single performance of the assay. In such cases, at leastsome (for example all) of the biomarkers may be measured at one time.Certain embodiments may provide, for example, systems and methods thatallow for the selective and sensitive detection and quantification ofbiomarkers (for example in a multiplex assay).

In many conventional assay systems, multiplexing may be hindered bynon-specific binding, cross-reactivity (for example between detectionmolecules for a first biomarker and a second biomarker), the relativeconcentrations of the biomarkers, and/or incompatibility between theassay conditions (for example dilution, detection molecules, pH,solvent, reagents) for a first biomarker and the assay conditions for asecond biomarker. For instance, certain conventional reagents necessaryfor ultrasensitive detection of a first analyte (for example Tau) maynot allow for accurate detection of a second analytes (for example UCHL1). For example, certain conventional detergents while beneficial forthe detection of Tau may induce conformational changes (for example dueto physicochemical alterations in solvation activity) in other analytes(for example biomarkers), such as UCH L1, that adversely affectpresentation of the portion (for example antigenic epitope) of theanalyte utilized for detection. For instance, a conventional detergentmay cause the antigenic epitope of an analyte to be less available oroverly available for binding to a detection molecule (for exampleantibody) resulting in inaccurate quantification and/or a relativelyhigh LOQ and/or LOD. As another example, certain detection moleculesutilized for ultrasensitive detection of a biomarker may cross-react ornon-specifically bind with another biomarker or detection moleculeresulting in inaccurate quantification of the biomarker. For instance,detection molecules for certain biomarkers (for example NF-L, GFAP,and/or Tau) may cross-react or non-specifically bind with UCH L1resulting in inaccurate quantification of the biomarker(s). As yetanother example, molecules used to calibrate the multiplex assay maynon-specifically bind resulting in inaccurate quantification. Forinstance, a biomarker standard (for example GFAP standard) used forcalibration of the biomarker may non-specifically bind to assaycomponents (for example molecules, beads) necessary for the detection(for example quantification) of other biomarkers.

In certain embodiments, for example, the concentration of detectionmolecules that affords relatively low LODs and LOQs in a non-multiplexassay may result in cross-reactivity or non-specific binding in amultiplex assay. In certain embodiments, for example, a high sensitivityassay may be provided which requires relatively low background levels,such that signal-to-noise may be adequate at low analyte (for examplebiomarker) concentrations to permit reliable measurement. Non-specificbinding between assay components for a biomarker and assay componentsfor another biomarker increases background levels resulting in a reducedsignal-to-noise ratio and accordingly reduced sensitivity. In certainembodiments, for example, certain conventional diluents that produceacceptable dilution linearity and spike recovery for the detection of abiomarker may result in dilution non-linearity and/or unpredictablespike recovery for another biomarker. In many assays, one or moreblocker reagents may be included in a sample diluent to mimic thephysicochemical properties of the native sample in order to allow foracceptable dilution linearity. Diluents, and accordingly the blockerreagents comprised therein, that do not result in linear dilution may beunsuitable for use in the assay. In multiplex assays, suitable diluentsshould produce acceptable dilution linearity for each analyte (forexample biomarkers). Many conventional diluents generally employed maybe unsuitable for multiplexing. The problems associated withmultiplexing often amplify as the number of biomarkers to be multiplexedincreases. Thus, for many conventional assay systems, multiplexing ofcertain biomarkers may be difficult and/or not possible.

Certain embodiments may provide, for example, methods, tests, assays,kits, or systems that do not suffer from one or more limitations ofconventional immunoassays with respect to multiplexing biomarkers, andthus can provide an improved way to measure biomarkers associated withbrain injury and/or neurodegeneration. In certain embodiments, forexample, the methods, tests, assays, kits, or systems provideultra-sensitive detection of two or more biomarkers selected from thegroup consisting of NF-L, GFAP, UCH L1, and Tau. In certain embodiments,for example, the methods, tests, assays, kits, or systems may comprisecertain assay conditions (for example detection molecules, blockers,detergents, concentrations) that allow for sub-femtomolar detection ofat least some (for example each) of the biomarkers.

In certain embodiments, for example, the methods, tests, assays, kits,or systems may utilize one or more reagents and/or techniques thatreduce and/or eliminate cross-reactivity, non-specific binding, dilutionnon-linearity, unpredictable spike recovery, and/or adverse analyte (forexample protein) confirmation. In certain embodiments, for example, themethods, tests, assays, kits, or systems may utilize a detergent thatpromotes favorable biomarker confirmation for at least some (for exampletwo or more, three or more, all) of the biomarkers in the multiplexassay. In certain embodiments, for example, the methods, tests, assays,kits, or systems may utilize a detergent that promotes a confirmation ofNF-L and another biomarker (for example UCH L1, GFAP, and/or Tau) thatmay be favorable for accurate detection (for example quantification). Incertain embodiments, for example, use of the detergents described hereinmay allow for the detection of multiple biomarkers in a single assaythat may not be measured using conventional detergents generallyemployed. In certain embodiments, for example, use of the detergentsdescribed herein may allow for the detection of both Tau and UCH-LI inthe multiplexed assay. Conversely, some conventional detergents thatallow for the detection of tau proteins cause UCH L1 to be undetectable.Non-limiting examples of suitable detergents include non-ionicdetergents, such as Triton™ X-100, the non-ionic surfactant sold underthe trademark Triton™ X-114, Tween-20, Tween-80, and combinationsthereof.

In certain embodiments, for example, the detergent may be used duringone or more assay steps and/or present in one or more assay compositions(for example diluent composition). In certain embodiments, for example,the detergent may be present during the detection step. In certainembodiments, for example, the detergent may be present in the diluentcomposition. In certain embodiments, for example, the methods, tests,assays, kits, or systems may utilize a diluent comprising a detergent(for example Triton™ X-100 or Triton™ X-114) at a concentration ofbetween about 0.1 wt. % and about 1.0 w.t % (for example about 0.5 wt.%). In certain embodiments, for example, the presence of the detergentin the diluent composition may allow for the accurate detection (forexample quantification) of the biomarkers in the multiplex assay and/ora relatively low LOD and/or LOQ. In certain embodiments, for example,the absence of a detergent in one or more assay steps and/or assaycompositions may result in one or more biomarkers (for example UCH L1)being undetectable.

In certain embodiments, for example, the methods, tests, assays, kits,or systems may utilize one or more reagents that reduce and/or eliminatecross-reactivity or non-specific binding between assay components (forexample detection molecules, calibration molecules, biomarker) for onebiomarker and assay components for another biomarker. In certainembodiments, for example, the methods, tests, assays, kits, or systemsmay utilize detection and/or calibration molecules that have relativelylow non-specific binding and/or cross-reactivity with other componentsin the multiplex assay. In certain embodiments, for example, use of thedetection and/or calibration molecules described herein allowed for thedetection of multiple biomarkers in a single assay that could not beaccurately measured, measured with acceptable clinical discrimination,and/or measured with low LOQ and/or LOD using certain conventionaldetection and/or calibration molecules. In certain embodiments, forexample, the methods, tests, assays, kits, or systems may utilizecertain combinations of UCH L1 clones (for example human recombinantprotein, UCH L1 clones 10D1 and 1033) that provide relatively highclinical discrimination, relatively low non-specific binding, andrelatively low cross-reactivity with other assay components (for exampleother biomarkers). Conversely, conventional combinations of UCH L1clones that provide relatively low LOD for UCH L1 may produce poorclinical discrimination for other biomarkers, have high non-specificbinding with assay components for other biomarkers, or otherwiseadversely affect the detection of other biomarkers in the multiplexassay. In certain embodiments, for example, the methods, tests, assays,kits, or systems may utilize certain GFAP standards (for example Hytestnative human GFAP) for calibration that do not cross-react and/ornon-specifically bind with the assay components for the NF-L, UCH L1,and/or Tau assays, whereby the cross-reactivity between the GFAPcalibration standard that results in a non-specific signal in non-GFAPassays (for example NF-L, UCH-L1, and/or Tau assays) with increasingdoses of GFAP antigen may be reduced and/or eliminated.

In certain embodiments, for example, the concentration of one or morebinding ligands used in the multiplex assay may be less than theconcentration utilized in a single biomarker assay. In certainembodiments, for example, the binding ligand may be a molecule,particle, or the like which specifically binds to or otherwisespecifically associates with a biomarker molecule to aid in thedetection of the biomarker molecule. In certain embodiments, forexample, the binding ligand may comprise an antibody for the biomarkerthat may be directly or indirectly detected. Other embodiments arecontemplated herein. In certain embodiments, for example, theconcentration of one or more binding ligands used in the multiplex assaymay range from about 2 to about 10 (for example from about 2 to about 8,from about 4 to about 10, from about 4 to about 8) times less than theconcentration utilized in a single biomarker assay. In certainembodiments, for example, the concentration of binding ligands used in asingle biomarker assay may result in cross-reactivity and/ornon-specific binding with assay components for another assay. In certainembodiments, for example, the UCH L1 binding ligand (for example clone1033) concentrations for a single biomarker assay may result in arelatively high amount of non-specific binding. In certain embodiments,for example, the concentration of the UCH L1 binding ligand in themultiplex assay may be at least about 2 times (for example at leastabout 4 times, about 6 times) less than the concentration utilized inthe single biomarker assay. In certain embodiments, for example, theconcentration of one or more binding ligands in the multiplex assay maybe in the range of from about 0.1 to about 2 pg/mL, whereby crossreactivity and/or non-specific binding may be reduced or eliminated.

In certain embodiments, for example, the methods, tests, assays, kits,or systems may utilize one or more reagents and/or techniques thatreduce and/or eliminate dilution non-linearity and unpredictable spikerecovery for each biomarker (for example NF-L, GFAP, UCH L1, and/orTau). In certain embodiments, for example, the methods, tests, assays,kits, or systems may utilize one or more blockers that promote dilutionlinearity (for example of a tested sample when diluted with a samplediluent) and predictable spike recovery. In certain embodiments, forexample, the one or more blockers may serve to reduce and/or eliminatesample matrix effects that adversely affect linearity, spike recovery,and/or quantification. In certain embodiments, for example, the term“blocker” may refer to a reagent (for example protein-containingreagent) designed to competitively block non-specific binding. Incertain embodiments, for example, the blocker may comprise one or moreproteins. In certain embodiments, for example, the blocker reagent maycontain multiple proteins (for example IgG), to block non-specificinteractions between monoclonal antibodies (for example mouse monoclonalantibodies). In certain embodiments, for example, the blocker reagentsmay comprise proteins or antibodies from different animal species (forexample mouse, bovine, human). Non-limiting examples of suitableblockers include globulin (for example bovine gamma globulin), albumin(for example BSA), TRU Block™ Superchemiblock™, and combinationsthereof. In certain embodiments, for example, the methods, tests,assays, kits, or systems may utilize (for example in a diluent) two ormore blockers (for example three or more, four). In certain embodiments,for example, a diluent may comprise a globulin (for example bovine gammaglobulin, in the range of about 0.005 wt. % to about 0.2 wt. %,) analbumin (for example BSA, in the range of about 0.01 wt. % to about0.1%), TRU Block™ (for example in the range of about 5 pg/mL to about 20pg/mL), and/or Superchemiblock™ (for example in the range of about 10pg/mL to about 100 pg/mL, in the range of about 10 pg/mL to about 100pg/mL). In certain embodiments, for example, the diluent may comprise acarbohydrate (for example dextrose, in a range of about 0.005 wt. % toabout 0.05 wt. %), a nitrogen containing small molecule (for exampleurea, in a range of about 1 mM to about 10 mM), and/or a detergent (forexample Triton™ X-100, in a range of about 0.2 wt. % to about 1 wt. %).

In certain embodiments, for example, the concentration of one or moreblockers used in the multiplex assay may be greater than theconcentration utilized in a single biomarker assay. In certainembodiments, for example, the concentration of one or more blockersand/or all blockers used in the multiplex assay may range from about 5to about 100 times (for example from about 10 to about 100, from about20 to about 100, from about 5 to about 50) greater than theconcentration utilized in a single biomarker assay. In certainembodiments, for example, the concentration of one or more blockers (forexample TRU™ block, Superchemiblock™) and/or all blockers in an assaycomposition (for example diluent, detection composition) may be at leastabout 10 pg/mL, at least about 15 pg/mL, at least about 20 pg/mL, atleast about 30 pg/mL, at least about 40 pg/mL, at least about 50 pg/mL,at least about 60 pg/mL, at least about 70 pg/mL, at least about 80pg/mL, at least about 90 pg/mL, at least about 100 pg/mL, at least about125 pg/mL, at least about 150 pg/mL, at least about 175 pg/mL, or atleast about 200 pg/mL. In certain embodiments, for example, theconcentration of one or more blockers (for example TRU™ block,Superchemiblock™) and/or all blockers in an assay composition may rangesfrom about 10 pg/mL to about 200 pg/mL (for example 10 pg/mL to about100 pg/mL, 5 pg/mL to about 20 pg/mL, 50 pg/mL to about 100 pg/mL).

Certain embodiments may provide, for example, methods, tests, assays,kits, or systems that are ultrasensitive and have very low limits ofquantification and/or limits of detection (for example in the low pg/mLrange). In certain embodiments, for example, the methods, tests, assays,kits, or systems may be used to provide statistically significant and/ormeaningful results regarding a measure of the concentration of thepanels of two or more biomarkers associated with brain injury and/orneurodegeneration. In certain embodiments, for example, the methods,tests, assays, kits, or systems may have a limit of detection and/or alimit of quantification of less than about or about 500 pg/mL, less thanabout or about 250 pg/mL, less than about or about 100 pg/mL, less thanabout or about 50 pg/mL, less than about or about 40 pg/mL, less thanabout or about 30 pg/mL, less than about or about 20 pg/mL, less thanabout or about 10 pg/mL, less than about or about 8 pg/mL, less thanabout or about 6 pg/mL, less than about or about 5 pg/mL, less thanabout or about 4 pg/mL, less than about or about 3 pg/mL, less thanabout or about 2 pg/mL, less than about or about 1 pg/mL, less thanabout or about 0.8 pg/mL, less than about or about 0.7 pg/mL, less thanabout or about 0.6 pg/mL, less than about or about 0.5 pg/mL, less thanabout or about 0.4 pg/mL, less than about or about 0.3 pg/mL, less thanabout or about 0.2 pg/mL, less than about or about 0.1 pg/mL, less thanabout or about 0.08 pg/mL, less than about or about 0.06 pg/mL, lessthan about or about 0.05 pg/mL, less than about or about 0.04 pg/mL,less than about or about 0.02 pg/mL, less than about or about 0.01pg/mL, or less than about or about 0.005 pg/mL for at least some (forexample each) of the biomarkers. In certain embodiments, for example,the methods, tests, assays, kits, or systems may have a limit ofquantification and/or a limit of detection between about 100 pg/mL andabout 0.01 pg/mL, between about 50 pg/mL and about 0.02 pg/mL, orbetween about 25 pg/mL and about 0.02 pg/mL, between about 10 pg/mL andabout 0.02 pg/mL for at least some (for example each) of the biomarkers.

In certain embodiments, for example, an LOQ and/or LOD may differ forone or more (for example each) of the biomarkers determined with thesame assay and/or when two or more biomarkers are determined together ina single assay and/or from a single sample. In certain embodiments, forexample, the LOD for Tau may be equal to or less than about 0.02 pg/mLand/or the LOQ for Tau may be equal to or less than about 0.1 pg/mL (forexample equal to or less than about 0.06 pg/mL), for example whenmeasured with one or more other biomarkers. In certain embodiments, forexample, the LOD for NF-L may be equal to or less than about 0.2 pg/mL(for example equal to or less than about 0.1 pg/mL) and/or the LOQ forNF-L may be equal to or less than about 1.0 pg/mL (for example equal toor less than about 0.4 pg/mL, equal to or less than about 0.3 pg/mL),for example when measured with one or more other biomarkers. In certainembodiments, for example, the LOD for GFAP may be equal to or less thanabout 0.3 pg/mL and/or the LOQ for GFAP may be equal to or less thanabout 1.0 pg/mL (for example equal to or less than about 0.5 pg/mL), forexample when measured with one or more other biomarkers. In certainembodiments, for example, the LOD for UCH L1 may be equal to or lessthan about 5 pg/mL (for example equal to or less than about 2 pg/mL)and/or the LOQ for UCH-L1 may be equal to or less than about 10 pg/mL(for example equal to or less than about 5 pg/mL), for example whenmeasured with one or more other biomarkers.

The terms “limit of detection” (or LOD) and “limit of quantification”(or LOQ) are given their ordinary meaning in the art. The LOD refers tothe lowest analyte concentration likely to be reliably distinguishedfrom background noise and at which detection is feasible. The LOD asused herein may be defined as three standard deviations (SD) abovebackground noise. The LOQ refers to the lowest concentration at whichthe analyte can not only be reliably detected but at which somepredefined goals for bias and imprecision are met. Generally, as is usedherein, the LOQ refers to the lowest concentration above the LOD whereinthe coefficient of variation (CV) of the measured concentrations lessthan about 20%.

In certain embodiments, for example, the methods, tests, assays, kits,or systems may comprise comparing the levels of the one or morebiomarkers with levels of the biomarkers obtained from a certainpopulation of individuals. In certain embodiments, for example, themethods, tests, assays, kits, or systems may comprise comparing thelevels of the one or more biomarkers with levels of the biomarkersobtained from a population of individuals having a certain gender, age,ethnicity, health status, disease, phenotype, and/or genotype. Incertain embodiments, for example, the methods, tests, assays, kits, orsystems may comprise comparing the levels of the one or more biomarkerswith levels of the biomarkers obtained from a population of healthyindividuals. In certain embodiments, for example, the methods, tests,assays, kits, or systems may comprise comparing the levels of the one ormore biomarkers with levels of the biomarkers obtained from a populationof individuals with a history of one/or more brain injury events. Incertain embodiments, for example, the methods, tests, assays, kits, orsystems may comprise collecting a sample from the patient (for example avenous or capillary blood sample). In certain embodiments, for example,the sample may be collected after a suspected brain injury. In certainembodiments, for example, the sample may be collected after an eventcreating a risk (for example a heightened risk) of brain injury (forexample child birth or detonation of an explosive) or an event prone tocausing brain injury. In certain embodiments, for example, the samplemay be collected within a certain timeframe of the brain injury orsuspected brain injury. In certain embodiments, for example, thetimeframe may be selected such that a measure of the concentration ofthe biomarkers in the sample becomes statistically significant. Incertain embodiments, for example, the period of time between the braininjury or suspected brain injury and collection of the blood sample fromthe patient may account for any lag time required for the biomarkers tocross the blood brain barrier (BBB). Non-limiting examples of suitableperiods of time in which a sample may be obtained from the patientinclude 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 8 hours, 10 hours,12 hours, 18 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 4days, 5 days, 6 days, 7 days, or more. In certain embodiments, forexample, the duration of time between suspected brain injury and samplecollection may be at least 60 hours or at least 72 hours. In certainembodiments, for example, the duration of time may be between 12 hoursand 7 days, between 24 hours and 4 days, between 2 days and 4 days, orbetween 3 days and 4 days. In certain embodiments, for example, thesample may be obtained from the patient within a short timeframefollowing the brain injury or suspected brain injury. For example, thesample may be obtained from the patient within 1 hour, 2 hours, 3 hours,4 hours, 5 hours, 6 hours, 8 hours, 10 hours, or 12 hours of the braininjury. In certain embodiments, for example, the sample may be obtainedwithin 6 hours of the brain injury. In certain embodiments, for example,the sample collection may occur months following the brain injury inorder to assess long-term effects. In certain embodiments, for example,the samples may be collected 14 days, 1 month, 3 month, 6 months, 9months, or more, following brain injury.

In certain embodiments, for example, the sample obtained from thepatient may be from any suitable bodily source. In certain embodiments,for example, the sample may be a CSF fluid sample. In certainembodiments, for example, the sample may be exclusive of CSF fluid. Incertain embodiments, for example, the sample may be blood (for examplevenous blood or capillary blood) or a blood product (for example wholeblood, plasma, serum, etc.). In certain embodiments, for example, thesample may be a urine or a saliva sample. In certain embodiments, forexample, the sample may be analyzed directly (for example without theneed for extraction of the biomarker from the fluid sample) and/or withdilution (for example addition of a buffer or agent to the sample).Those of ordinary skill in the art will be aware of suitable systems andmethods for obtaining a sample from a patient. In addition to thebiomarkers specifically mentioned herein, those of ordinary skill willbe aware of other suitable biomarkers to use in connection with themethods described herein. In certain embodiments, for example,biomarkers detected and/or quantified by the methods, tests, assays,kits, or systems may comprise, without limitation, neuron specificneuronal enolase (NSE), β-site aPP-cleaving enzyme 1 (BACe1), S100B,myelin basic protein (MBP), growth associated protein 43, glutaminesynthetase, GFAP, glycine transporter (for example GLYT1, GLYT2), neuronspecific glycoprotein (for example GP50), calpain, neurofibrillaryprotein, heat shock protein 72, beta-amyloid precursor proteins,calbindin D-28K, proteolipid protein, myeline associated glycoprotein,neurofilament H, creatine kinase protein (for example CK-BB), tauproteins (including phosphorylated taus such as p-tau-81 or p-tau-231),and endothelium membrane proteins (for example thrombomodulin).

In certain embodiments, for example, the methods, tests, assays, kits,or systems may comprise detecting and/or quantifying a panel ofbiomarkers. In certain embodiments, for example, the panel of biomarkersmay include at least one species of tau protein. Various forms and/orcombinations of tau proteins are contemplated for use as a targetbiomarker with the methods described herein, including isoforms andshort isoforms, for example, ranging from tau 23 (352a.a, “0N3R”,wherein R indicates the number of repeats and N indicates the number oramino terminal inserts, as will be understood by those of ordinary skillin the art) to tau 40 (441a.a, “2N4R”). There are six knownnaturally-occurring tau proteins, the sequences of which are well knownin the art. The six tau proteins include tau 23 (352, 0N3R), tau 24(383, 0N4R), tau 37 (381, 1N3R), tau 34 (412, 1N4R), tau 39 (410, 2N3R),tau 40 (441, 2N4R), and/or combinations thereof. In certain embodiments,for example, at least some of the tau proteins may be phosphorylated.

Those of ordinary skill in the art will understand that determination ofa biomarker in a sample may comprise determining a measure of theconcentration a single isoform of a biomarker, or alternatively, maycomprise determining a measure of the concentration of a plurality (forexample 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10) of isoforms of thebiomarker. In certain embodiments, for example, a measure of theconcentration of tau protein employed in the methods described hereinmay be a measure of the concentration of a single isoform of tau proteinin the sample, or alternatively, a measure of the concentration of tauprotein employed in the algorithms and methods described herein may be ameasure of the concentration of a plurality of forms of tau proteins inthe sample.

In certain embodiments, for example, the methods, tests, assays, kits,or systems may comprise using an immunoassay to determine a measure ofthe concentration of two or more biomarkers. In certain embodiments, forexample, the immunoassay may utilize an antibody as a detectionmolecule. In general, any suitable antibodies that differentially and/orselectively binds a biomarker may be used.

An immunoassay is a biochemical test that measures the presence orconcentration of a molecule of interest (for example a macromoleculesuch as a protein) in a sample through the use of an antibody orimmunoglobulin. Typically, an antibody specific to the molecule ofinterest interacts with the molecule in an immunoassay. The antibody canbe labeled, directly or indirectly such that those bound to the moleculecould release a detectable signal. Presence or concentration of themolecule of interest can be determined based on the level of thedetectable signal. Certain embodiments may provide, for example, animmunoassay using one or more types of labels including, withoutlimitation, enzymes, radioactive isotopes, DNA reporters, fluorogenicreporters, electrochemiluminescent tags, all of which are well known inthe art. In certain embodiments, for example, the immunoassay mayamplify a signal via a catalyst (for example an enzyme). In certainembodiments, for example, the immunoassay may be exclusive of a label.

In certain embodiments, for example, the immunoassay may be used todetect an antigen of interest (for example NF-L, tau protein, GFAP, UCHL1, etc.). In certain embodiments, for example, the immunoassay maycomprise a protocol that comprises certain standard techniques known inthe art. In certain embodiments, for example, the immunoassay may be acompetitive immunoassay. In certain embodiments, for example, theimmunoassay may be a one-site non-competitive assay. In certainembodiments, for example, the immunoassay may be a two-sitenoncompetitive assay (for example a sandwich assay). In certainembodiments, for example, the immunoassay may comprise multiple stepswith reagents being added and washed away or separated at differentpoints in the assay (for example a heterogeneous immunoassay). Incertain embodiments, for example, the immunoassay may be carried outsimply by mixing the reagents and sample and making a physicalmeasurement (for example a homogenous immunoassay).

In a preferred embodiment, for example, the immunoassay may be anenzyme-linked immunosorbent assay (ELISA). In certain embodiments, forexample, the immunoassay may be a digital assay (for example a digitalELISA). In certain embodiments, for example, the digital ELISA mayincorporate single molecule array technology (for example digitalimmunoassay technology sold under the Simoa trademark) as describedherein. Additional details regarding single molecule array technologiesare described herein.

The basic nature of the ELISA format is generally well known in the art.The inventive ELISA type assays used in certain embodiments of thedetection methods described herein can incorporate a variety of formatsknown in the art, including direct ELISA, Sandwich ELISA, competitiveELISA, and multiple and ready-to-use ELISA. In a typical “indirect”ELISA, an antibody having specificity for the antigen of interest isimmobilized on a solid surface (for example the wells of a standardmicrotiter assay plate, or the surface of a microbead or a microarray)and a sample comprising, for example bodily fluid or substancesextracted from stool samples, to be tested for the presence of theantigen is brought into contact with the immobilized antibody. Anyantigen of interest in the sample will bind to the immobilized antibody.The bound antibody/antigen complexes may then be detected using anysuitable method. In one embodiment, a second antibody, whichspecifically recognizes an epitope of the antigen, which may bedifferent from the epitope recognized by the immobilized antibody, isused to detect the antibody/antigen complexes. The second antibody maybe usually labelled with a detectable marker (directly or indirectly).In some examples, the maker can be an enzyme such as peroxidase,alkaline phosphatase, or galactosidase, allowing quantitative detectionby the addition of a substrate for the enzyme which generates adetectable product, for example a colored, chemiluminescent orfluorescent product. Other types of detectable labels known in the artmay be used with equivalent effect. In other examples, the secondantibody may be labeled with a member of a receptor/ligand pair, forexample, biotin. An enzyme conjugate comprising an enzyme conjugatedwith the other member of the receptor/ligand pair, for examplestreptavidin, can be brought into contact with the second antibody. Asubstrate of the enzyme may be then added to produce a product thatreleases a detectable signal.

Generally, the methods employed have low limits of detection and/orlimits of quantification as compared to bulk analysis techniques (forexample ELISA methods). The use of assay methods that have low limits ofdetection and/or limits of quantification allows for correlations to bemade between the various parameters discussed above and a method oftreatment and/or diagnostic indication that may otherwise not bedeterminable and/or apparent.

An antibody (interchangeably used in plural form) may be animmunoglobulin molecule capable of specific binding to a target, such asNF-L, Tau, GFAP, and UCH L1, through at least one antigen recognitionsite, located in the variable region of the immunoglobulin molecule. Asused herein, the term “antibody” encompasses not only intact (i.e.,full-length) polyclonal or monoclonal antibodies, but alsoantigen-binding fragments thereof (such as Fab, Fab′, F(ab′)2, Fv),single chain (scFv), mutants thereof, fusion proteins comprising anantibody portion, humanized antibodies, chimeric antibodies, diabodies,linear antibodies, single chain antibodies, multispecific antibodies(for example bispecific antibodies) and any other modified configurationof the immunoglobulin molecule that comprises an antigen recognitionsite of the required specificity, including glycosylation variants ofantibodies, amino acid sequence variants of antibodies, and covalentlymodified antibodies. An antibody includes an antibody of any class, suchas IgD, IgE, IgG, IgA, or IgM (or sub-class thereof), and the antibodyneed not be of any particular class. Depending on the antibody aminoacid sequence of the constant domain of its heavy chains,immunoglobulins can be assigned to different classes. There are fivemajor classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, andseveral of these may be further divided into subclasses (isotypes), forexample IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy-chain constantdomains that correspond to the different classes of immunoglobulins arecalled alpha, delta, epsilon, gamma, and mu, respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known. The antibodies described herein can bemurine, rat, human, or any other origin (including chimeric or humanizedantibodies).

In certain embodiments, for example, the analyte antibodies describedherein may have a suitable binding affinity to the antigen. As usedherein, “binding affinity” refers to the apparent association constantor K_(A). The K_(A) is the reciprocal of the dissociation constant(K_(D)). The antibody described herein may have a binding affinity(K_(D)) of at least 10⁻⁵, 10⁻⁶, 10⁻⁷, 10⁻⁸, 10⁻⁹, 10⁻¹⁰ M, or lower. Anincreased binding affinity corresponds to a decreased K_(D). Higheraffinity binding of an antibody to a first target relative to a secondtarget can be indicated by a higher K_(A) (or a smaller numerical valueK_(D)) for binding the first target than the K_(A) (or numerical valueK_(D)) for binding the second target. In such cases, the antibody hasspecificity for the first target relative to the second target.Differences in binding affinity (for example for specificity or othercomparisons) can be at least 1.5, 2, 3, 4, 5, 10, 15, 20, 37.5, 50, 70,80, 91, 100, 500, 1000, 10,000 or 10⁵ fold.

Binding affinity can be determined by a variety of methods includingequilibrium dialysis, equilibrium binding, gel filtration, ELISA,surface plasmon resonance, or spectroscopy (for example using afluorescence assay). Exemplary conditions for evaluating bindingaffinity are in HBS-P buffer (10 mM HEPES pH7.4, 150 mM NaCl, 0.005%(v/v) Surfactant P20). These techniques can be used to measure theconcentration of bound binding protein as a function of target proteinconcentration. The concentration of bound binding protein ([Bound]) maybe related to the concentration of free target protein ([Free]) and theconcentration of binding sites for the binding protein on the targetwhere (N) may be the number of binding sites per target molecule by thefollowing equation:

[Bound]=[N][Free]/(K _(D)+[Free])

It is not always necessary to make an exact determination of K_(A),though, since sometimes it may be sufficient to obtain a quantitativemeasurement of affinity, for example determined using a method such asELISA or FACS analysis, which is proportional to K_(A), and thus can beused for comparisons, such as determining whether a higher affinity is,for example 2-fold higher, to obtain a qualitative measurement ofaffinity, or to obtain an inference of affinity, for example by activityin a functional assay, for example an in vitro or in vivo assay.

In certain embodiments, for example, the antibodies used in thedetection assays described herein may differentially bind one biomarkerassociated with brain injury and/or neurodegeneration over another suchbiomarker. An antibody that “differentially binds” to a first target ora first epitope as relative to a second target or a second epitoperefers to an antibody that has different binding affinities to the firstand second targets or different binding affinities to the first andsecond epitopes. In certain embodiments, for example, an antibody mayhave a much higher binding affinity to the first target/epitope asrelative to the second target/epitope, or vice versa, for example atleast 2-fold higher, 5-fold higher, 10-fold higher, 50-fold higher,100-fold higher, 200-fold higher, 500-fold higher, 1,000-fold higher, or10,000-fold higher. In other examples, the antibody may have a muchlower binding affinity to the first target/epitope as relative to thesecond target/epitope, or vice versa, for example at least 2-fold lower,5-fold lower, 10-fold lower, 50-fold lower, 100-fold lower, 200-foldlower, 500-fold lower, 1,000-fold lower, or 10,000-fold lower.

In certain embodiments, for example, the antibodies used in thedetection assays described herein may specifically bind one biomarkerassociated with brain injury and/or neurodegeneration over another suchbiomarker. An antibody “specifically binds” to a target antigen if itbinds with greater affinity, avidity, more readily, and/or with greaterduration than it binds to other substances. It is also understood byreading this definition that, for example, an antibody that specificallybinds to a first target antigen may or may not specifically orpreferentially bind to a second target antigen. As such, “specificbinding” or “preferential binding” does not necessarily require(although it can include) exclusive binding. Generally, but notnecessarily, reference to binding means preferential binding.

Antibodies capable of binding biomarkers associated with brain injuryand/or neurodegeneration can be made by any method known in the artand/or are commercially available. See, for example, Harlow and Lane,(1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,New York.

In certain embodiments, for example, the methods, tests, assays, kits,or systems may comprise determining a measure of the concentration of apanel of at least one biomarker in one or more samples obtained from apatient following a suspected brain injury. In certain embodiments, forexample, a diagnosis, prognostic indication of the patient's recovery,and/or determining a course of treatment may be based at least in parton the measure of the concentration of the at least one biomarkerpresent in the one or more samples. In certain embodiments, for example,following determining the measure of the concentration of thebiomarkers, the measure of the concentration may be compared with apredefined level of the biomarker from a population of healthyindividuals. In certain embodiments, for example, the methods, tests,assays, kits, or systems may comprise comparing the measure of theconcentration of NF-L and at least one other biomarker (for example oneor more of GFAP, UCH L1, and Tau) with levels of NF-L and the at leastone other biomarker (for example levels based on measurements taken fromsamples obtained from a population of healthy individuals).

Certain embodiments may provide, for example, kits for use indetermining a measure of the concentration of at least one biomarker ina sample. In certain embodiments, for example, the at least onebiomarker may be at least two biomarkers, wherein a first biomarker ofthe at least two biomarkers may be NF-L and at least a second of the atleast two biomarkers may be at least one other biomarker selected fromthe group consisting of GFAP, UCH L1, and Tau. In certain embodiments,for example, the kit may be for measuring concentration of a biomarkerpanel comprising at least Tau, GFAP, UCH L1, and NF-L (for examplecomprising GFAP, UCH L1, and Tau). In certain embodiments, for example,the kit may comprise a plurality of capture objects (for example beads,optionally magnetic beads), each having a binding surface comprising aplurality of capture components. In certain embodiments, for example,the plurality of capture components may comprise a plurality of anantibodies having specific affinity for the one or more biomarkers beingdetected. In certain embodiments, for example, each capture object (forexample bead) may comprise a plurality of types of capture objects, eachtype of capture object having specific affinity for a particularbiomarker (for example a plurality of antibodies having specificaffinity for NF-L and a plurality of antibodies having specific affinityfor at least one other biomarker selected from the group consisting of,GFAP, UCH L1, and Tau). In certain embodiments, for example, the kit maycomprise a plurality of types of binding ligands having specificaffinity for the at least one biomarker. In certain embodiments, forexample, the binding ligands may be directly or indirectly detectable.In certain embodiments, for example, the kit may comprise a plurality ofa first type of binding ligand having affinity for NF-L and a pluralityof a second type of binding ligand having affinity for at least oneother biomarker selected from the group consisting of, GFAP, UCH L1, andTau. In certain embodiments, for example, the kit may comprise i) aplurality of capture objects, each having a binding surface comprising aplurality of capture components; ii) a plurality of a first type ofbinding ligand having affinity for NF-L; and iii) a plurality of asecond type of binding ligand and a third type of binding ligand havingaffinity for at least two other biomarker selected from the groupconsisting of, GFAP, UCH L1, and Tau. In certain embodiments, forexample, the kit may comprise i) a plurality of capture objects, eachhaving a binding surface comprising a plurality of capture components;ii) a plurality of a first type of binding ligand having affinity forNF-L; iii) a plurality of a second type of binding ligand havingaffinity for GFAP; iv) a plurality of a third type of binding ligandhaving affinity for UCH L1; and v) a plurality of a fourth type ofbinding ligand having affinity for Tau. In certain embodiments, forexample, the kit may comprise one or more components for performing theassays. In certain embodiments, for example, the kit may comprise anenzyme label substrate for indirect detection of a binding ligand. Incertain embodiments, for example, the kit may comprise an instructionmanual providing guidance for using the kit to perform any one of thedetection assay provided herein.

Exemplary Assay Methods and Systems

Certain embodiments, for example, may provide methods, tests, assays,kits, or systems having low limits of detection and/or limits ofquantification as compared to bulk analysis techniques (for examplemodified ELISA methods). In certain embodiments, for example, use of theprovided methods, tests, assays, kits, or systems may enable one or moremethods of treatment and/or diagnostic indication that may otherwise notbe determinable and/or apparent.

In certain embodiments, for example, a measure of the concentration ofbiomarker molecules in the fluid sample that may be substantiallyaccurately determined may be less than about or about 5000 fM, less thanabout or about 3000 fM, less than about or about 2000 fM, less thanabout or about 1000 fM, less than about or about 500 fM, less than aboutor about 300 fM, less than about or about 200 fM, less than about orabout 100 fM, less than about or about 50 fM, less than about or about25 fM, less than about or about 10 fM, less than about or about 5 fM,less than about or about 2 fM, less than about or about 1 fM, less thanabout or about 0.5 fM, less than about or about 0.1 fM, or less. Incertain embodiments, for example, a measure of the concentration ofbiomarker molecules in the fluid sample that may be substantiallyaccurately determined may be between about 5000 fM and about 0.1 fM,between about 3000 fM and about 0.1 fM, between about 1000 fM and about0.1 fM, between about 1000 fM and about 1 fM, between about 100 fM andabout 1 fM, between about 100 fM and about 0.1 fM, or the like. Incertain embodiments, for example, a measure of the concentration ofanalyte molecules or particles in a fluid sample may be considered to besubstantially accurately determined if the measured concentration of thebiomarker molecules in the fluid sample may be within about 10% of theactual (for example true) concentration of the biomarker molecules inthe fluid sample. In certain embodiments, for example, the measuredconcentration of the biomarker molecules in the fluid sample may bewithin about 5%, within about 4%, within about 3%, within about 2%,within about 1%, within about 0.5%, within about 0.4%, within about0.3%, within about 0.2% or within about 0.1%, of the actualconcentration of the biomarker molecules in the fluid sample. In certainembodiments, for example, a measure of the concentration determined maydiffer from the true (for example actual) concentration by no greaterthan about 20%, no greater than about 15%, no greater than 10%, nogreater than 5%, no greater than 4%, no greater than 3%, no greater than2%, no greater than 1%, or no greater than 0.5%. In certain embodiments,for example, the accuracy of the assay method may be determined bydetermining a measure of the concentration of biomarker molecules in afluid sample of a known concentration using the selected assay method.

Certain embodiments may provide, for example, an assay methodcomprising: spatially segregating biomarker molecules into a pluralityof locations to facilitate detection/quantification, such that eachlocation comprises/contains either zero or one or more biomarkermolecules. In certain embodiments, for example, the locations may beconfigured in a manner such that each location can be individuallyaddressed. In certain embodiments, for example, a measure of theconcentration of biomarker molecules in a fluid sample may be determinedby detecting biomarker molecules immobilized with respect to a bindingsurface having affinity for at least one type of biomarker molecule. Incertain embodiments, for example, the binding surface may form (forexample a surface of a well/reaction vessel on a substrate) or becontained within (for example a surface of a capture object, such as abead, contained within a well) one of a plurality of locations (forexample a plurality of wells/reaction vessels) on a substrate (forexample plate, dish, chip, optical fiber end, etc.). In certainembodiments, for example, at least a portion of the locations may beaddressed and a measure indicative of the number/percentage/fraction ofthe locations containing at least one biomarker molecule may be made. Incertain embodiments, for example, based upon thenumber/percentage/fraction, a measure of the concentration of biomarkermolecules in the fluid sample may be determined. In certain embodiments,for example, the measure of the concentration of biomarker molecules inthe fluid sample may be determined by a digital analysis method/system.In certain embodiments, for example, the measure of the concentration ofbiomarker molecules in the fluid sample may be determined by a digitalanalysis method/system employing Poisson distribution adjustment. Incertain embodiments, for example, the measure of the concentration ofbiomarker molecules in the fluid sample may be determined by a digitalanalysis method/system based at least in part on a measured intensity ofa signal. In certain embodiments, for example, the assay method (orapparatus or systems performing at least a portion of the assay method)may be automated.

Any of the methods, tests, assays, kits, or systems may employ one ormore of the methods and systems for spatially segregating analytemolecules (for example biomarkers) described in U.S. Patent ApplicationPublication No. US-2007-0259448 (Ser. No. 11/707,385), filed Feb. 16,2007, entitled “METHODS AND ARRAYS FOR TARGET ANALYTE DETECTION ANDDETERMINATION OF TARGET ANALYTE CONCENTRATION IN SOLUTION,” by Rissin etal.; U.S. Patent Application Publication No. US-2007-0259385 (Ser. No.11/707,383), filed Feb. 16, 2007, entitled “METHODS AND ARRAYS FORDETECTING CELLS AND CELLULAR COMPONENTS IN SMALL DEFINED VOLUMES,” byRissin et al.; U.S. Patent Application Publication No. US-2007-0259381(Ser. No. 11/707,384), filed Feb. 16, 2007, entitled “METHODS AND ARRAYSFOR TARGET ANALYTE DETECTION AND DETERMINATION OF REACTION COMPONENTSTHAT AFFECT A REACTION,” by Rissin et al.; International PatentPublication No. WO 2009/029073 (International Patent Application No.PCT/US2007/019184), filed Aug. 30, 2007, entitled “METHODS OFDETERMINING THE CONCENTRATION OF AN ANALYTE IN SOLUTION,” by Walt etal.; U.S. Patent Application Publication No. US-2010-0075862 (Ser. No.12/236,484), filed Sep. 23, 2008, entitled “HIGH SENSITIVITYDETERMINATION OF THE CONCENTRATION OF ANALYTE MOLECULES OR PARTICLES INA FLUID SAMPLE,” by Duffy et al.; U.S. Patent Application PublicationNo. US-2010-0075407 (Ser. No. 12/236,486), filed Sep. 23, 2008, entitled“ULTRA-SENSITIVE DETECTION OF MOLECULES ON SINGLE MOLECULE ARRAYS,” byDuffy et al.; U.S. Patent Application Publication No. US-2010-0075439(Ser. No. 12/236,488), filed Sep. 23, 2008, entitled “ULTRA-SENSITIVEDETECTION OF MOLECULES BY CAPTURE-AND-RELEASE USING REDUCING AGENTSFOLLOWED BY QUANTIFICATION,” by Duffy et al.; International PatentPublication No. WO2010/039179 (International Patent Application No.PCT/US2009/005248), filed Sep. 22, 2009, entitled “ULTRA-SENSITIVEDETECTION OF MOLECULES OR ENZYMES,” by Duffy et al.; U.S. PatentApplication Publication No. US-2010-0075355 (Ser. No. 12/236,490), filedSep. 23, 2008, entitled “ULTRA-SENSITIVE DETECTION OF ENZYMES BYCAPTURE-AND-RELEASE FOLLOWED BY QUANTIFICATION,” by Duffy et al.; U.S.patent application Ser. No. 12/731,130, filed Mar. 24, 2010, publishedas US-2011-0212848 on Sep. 1, 2011, entitled “ULTRA-SENSITIVE DETECTIONOF MOLECULES OR PARTICLES USING BEADS OR OTHER CAPTURE OBJECTS,” byDuffy et al.; International Patent Application No. PCT/US2011/026645,filed Mar. 1, 2011, published as WO 2011/109364 on Sep. 9, 2011,entitled “ULTRA-SENSITIVE DETECTION OF MOLECULES OR PARTICLES USINGBEADS OR OTHER CAPTURE OBJECTS,” by Duffy et al.; International PatentApplication No. PCT/US2011/026657, filed Mar. 1, 2011, published as WO2011/109372 on Sep. 9, 2011, entitled “ULTRA-SENSITIVE DETECTION OFMOLECULES USING DUAL DETECTION METHODS,” by Duffy et al.; U.S. patentapplication Ser. No. 12/731,135, filed Mar. 24, 2010, published asUS-2011-0212462 on Sep. 1, 2011, entitled “ULTRA-SENSITIVE DETECTION OFMOLECULES USING DUAL DETECTION METHODS,” by Duffy et al.; InternationalPatent Application No. PCT/US2011/026665, filed Mar. 1, 2011, publishedas WO 2011/109379 on Sep. 9, 2011, entitled “METHODS AND SYSTEMS FOREXTENDING DYNAMIC RANGE IN ASSAYS FOR THE DETECTION OF MOLECULES ORPARTICLES,” by Rissin et al.; U.S. patent application Ser. No.12/731,136, filed Mar. 24, 2010, published as US-2011-0212537 on Sep. 1,2011, entitled “METHODS AND SYSTEMS FOR EXTENDING DYNAMIC RANGE INASSAYS FOR THE DETECTION OF MOLECULES OR PARTICLES,” by Duffy et al.;U.S. patent application Ser. No. 13/035,472, filed Feb. 25, 2011,published as US 2012-0196774, entitled “SYSTEMS, DEVICES, AND METHODSFOR ULTRA-SENSITIVE DETECTION OF MOLECULES OR PARTICLES,” by Fournier etal.; or U.S. patent application Ser. No. 13/037,987, filed Mar. 1, 2011,published as US-2011-0245097 on Oct. 6, 2011, entitled “METHODS ANDSYSTEMS FOR EXTENDING DYNAMIC RANGE IN ASSAYS FOR THE DETECTION OFMOLECULES OR PARTICLES,” by Rissin et al.; each herein incorporated byreference.

Certain embodiments may provide, for example, methods, tests, assays,kits, or systems comprising spatially segregating biomarker moleculesfor detecting and/or quantifying the biomarker molecules (for example ina sample). In certain embodiments, for example, the methods, tests,assays, kits, or systems may comprise immobilizing a plurality ofbiomarker molecules with respect to a plurality of capture objects (forexample beads) that each include a binding surface having affinity forat least one type of biomarker. In certain embodiments, for example, thecapture objects may comprise a plurality of beads comprising a pluralityof capture components (for example an antibody having specific affinityfor a biomarker of interest, etc.). In certain embodiments, for example,at least a portion of the capture objects (for example at least aportion that is associated with at least one biomarker molecule) may bespatially separated/segregated into a plurality of locations, and atleast some of the locations may be addressed/interrogated (for exampleusing an imaging system). In certain embodiments, for example, a measureof the concentration of biomarker molecules in the fluid sample may bedetermined based on the information received when addressing thelocations (for example using the information received from the imagingsystem and/or processed using a computer implemented control system). Incertain embodiments, for example, a measure of the concentration may bebased at least in part on the number of locations determined to containa capture object that is or was associated with at least one biomarkermolecule. In certain embodiments, for example, a measure of theconcentration may be based at least in part on an intensity level of atleast one signal indicative of the presence of a plurality of biomarkermolecules and/or capture objects associated with a biomarker molecule atone or more of the addressed locations.

In certain embodiments, for example, a number/percentage/fraction oflocations containing a capture object but not containing a biomarkermolecule may also be determined and/or the number/percentage/fraction oflocations not containing any capture object may also be determined. Incertain embodiments, for example, a measure of the concentration ofbiomarker molecules in the fluid sample may be based at least in part ona ratio of the number of locations determined to contain a captureobject associated with a biomarker molecule to the total number oflocations determined to contain a capture object not associated with abiomarker molecule. In certain embodiments, for example, a measure ofthe concentration of biomarker molecules in the fluid sample a measureof the concentration of biomarker molecule in the fluid sample may bebased at least in part on the ratio of the number of locationsdetermined to contain a capture object associated with a biomarkermolecule to the number of locations determined to not contain anycapture objects. In certain embodiments, for example, a measure of theconcentration of biomarker molecules in the fluid sample, a measure ofthe concentration of biomarker molecule in the fluid sample may be basedat least in part on the ratio of the number of locations determined tocontain a capture object associated with a biomarker molecule to thenumber of locations determined to contain a capture object. In certainembodiments, for example, a measure of the concentration of biomarkermolecules in the fluid sample, a measure of the concentration ofbiomarker molecule in the fluid sample may be based on a combination oftwo or more of the foregoing measures. In certain embodiments, forexample, a measure of the concentration of biomarker molecules in thefluid sample, a measure of the concentration of biomarker molecule inthe fluid sample may be based may be based at least in part on the ratioof the number of locations determined to contain a capture object and abiomarker molecule to the total number of locations addressed and/oranalyzed. In certain embodiments, for example, a measure of theconcentration of biomarker molecules in the fluid sample, a measure ofthe concentration of biomarker molecule in the fluid sample may be basedon a combination of two or more of the foregoing measures.

In certain embodiments, for example, at least some of the plurality ofcapture objects (for example at least some associated with at least onebiomarker molecule) may be spatially separated into a plurality oflocations, for example, a plurality of reaction vessels in an arrayformat. The plurality of reaction vessels may be formed in, on and/or ofany suitable material. In certain embodiments, for example, the reactionvessels may be sealed or may be formed upon the mating of a substratewith a sealing component. In certain embodiments, for example, (forexample where quantization of the capture objects associated with atleast one biomarker molecule is desired), the partitioning of thecapture objects may be performed such that at least some (for example astatistically significant fraction; for example as described inInternational Patent Application No. PCT/US2011/026645, filed Mar. 1,2011, published as WO 2011/109364 on Sep. 9, 2011, entitled“ULTRA-SENSITIVE DETECTION OF MOLECULES OR PARTICLES USING BEADS OROTHER CAPTURE OBJECTS,” by Duffy et al., herein incorporated byreference) of the reaction vessels comprise at least one or, in certaincases, only one capture object associated with at least one biomarkermolecule and at least some (for example a statistically significantfraction) of the reaction vessels comprise an capture object notassociated with any biomarker molecules. In certain embodiments, forexample, the capture objects associated with at least one biomarkermolecule may be quantified, thereby allowing for the detection and/orquantification of biomarker molecules in the fluid sample by any of thetechniques described herein.

Certain embodiments may provide, for example, an assay method, wherein:a sample fluid containing or suspected of containing biomarker moleculesis provided; and an assay consumable comprising a plurality of assaysites is exposed to the sample fluid. In certain embodiments, forexample, the biomarker molecules may be provided in a manner (forexample at a concentration) such that a statistically significantfraction of the assay sites contain a single biomarker molecule and astatistically significant fraction of the assay sites do not contain anybiomarker molecules. In certain embodiments, for example, the assaysites may be exposed to a variety of reagents (for example using areagent loader) and or rinsed. In certain embodiments, for example, theassay sites may then optionally be sealed and imaged (see, for example,U.S. patent application Ser. No. 13/035,472, filed Feb. 25, 2011,published as US 2012-0196774, entitled “SYSTEMS, DEVICES, AND METHODSFOR ULTRA-SENSITIVE DETECTION OF MOLECULES OR PARTICLES,” by Fournier etal.). In certain embodiments, for example, the images may be analyzed(for example using a computer implemented control system) such that ameasure of the concentration of the biomarker molecules in the fluidsample may be obtained, based at least in part, by determination of thenumber/fraction/percentage of assay sites which contain a biomarkermolecule and/or the number/fraction/percentage of sites which do notcontain any biomarkers molecules. In certain embodiments, for example,the biomarker molecules may be provided in a manner (for example at aconcentration) such that at least some assay sites comprise more thanone biomarker molecule. In certain embodiments, for example, a measureof the concentration of biomarker molecules in the fluid sample may beobtained at least in part on an intensity level of at least one signalindicative of the presence of a plurality of biomarkers molecules at oneor more of the assay sites.

In certain embodiments, for example, the methods, tests, assays, kits,or systems may comprise exposing the fluid sample to a plurality ofcapture objects (for example beads). In certain embodiments, forexample, at least a portion of the biomarker molecules may beimmobilized with respect to a bead. In certain embodiments, for example,the biomarker molecules may be provided in a manner (for example at aconcentration) such that a statistically significant fraction of thebeads associate with a single biomarker molecule and a statisticallysignificant fraction of the beads do not associate with any biomarkermolecules. In certain embodiments, for example, at least a portion ofthe plurality of beads (for example those associated with a singlebiomarker molecule or not associated with any biomarker molecules) maythen be spatially separated/segregated into a plurality of assay sites(for example of an assay consumable). In certain embodiments, forexample, the assay sites may optionally be exposed to a variety ofreagents and/or rinsed. In certain embodiments, for example, at least aportion of the assay sites may then be addressed to determine the numberof assay sites containing a biomarker molecule. In certain embodiments,for example, the number of assay sites containing a bead not associatedwith a biomarker molecule, the number of assay sites not containing abead and/or the total number of assay sites addressed may also bedetermined. In certain embodiments, for example, the determined totalnumber of assay sites may then be used to determine a measure of theconcentration of biomarker molecules in the fluid sample. In certainembodiments, for example, more than one biomarker molecule may associatewith a bead and/or more than one bead may be present in an assay site.In certain embodiments, for example, the plurality biomarker moleculesmay be exposed to at least one additional reaction component prior to,concurrent with, and/or following spatially separating at least some ofthe biomarker molecules into a plurality of locations.

In certain embodiments, for example, the biomarker molecules may bedirectly detected or indirectly detected. In certain embodiments, forexample, one or more of the biomarker molecules may be directly detectedvia a molecule or moiety that is directly interrogated and/or detected(for example by one or more fluorescent entities attached to the one ormore biomarker molecules). In certain embodiments, for example, one ormore of the biomarker molecules may be indirectly detected by thepresence of an additional component. In certain embodiments, forexample, the biomarker molecules (for example optionally associated witha bead) may be exposed to at least one type of binding ligand. Incertain embodiments, for example, the at least one type of bindingligand may be adapted to be directly detected (for example the at leastone type of binding ligand may comprise a detectable molecule or moiety)or may be adapted to be indirectly detected (for example the at leastone type of binding ligand may including a component that can convert aprecursor labeling agent into a labeling agent). In certain embodiments,for example, a component of the at least one type of binding ligand maybe adapted to be directly detected via a measurable property (forexample a fluorescence emission, a color, etc.). In certain embodiments,for example, a component a component of the at least one type of bindingligand may facilitate indirect detection, for example, by converting aprecursor labeling agent into a labeling agent (for example an agentthat is detected in an assay). A “precursor labeling agent” is anymolecule, particle, or the like, that can be converted to a labelingagent upon exposure to a suitable converting agent (for example anenzymatic component). A “labeling agent” is any molecule, particle, orthe like, that facilitates detection, by acting as the detected entity,using a chosen detection technique. In certain embodiments, for example,the at least one type of binding ligand may comprise an enzymaticcomponent (for example horseradish peroxidase, beta-galactosidase,alkaline phosphatase, etc.). In certain embodiments, for example, afirst type of binding ligand may or may not be used in conjunction withone or more additional types of binding ligands (for example secondtype, etc.).

In certain embodiments, for example, the at least one type of bindingligand may comprise a plurality of types of binding ligands (for examplea first type of binding ligand and at least a second type of bindingligand). In certain embodiments, for example, a first type of bindingligand may be configured to associate with a first type of biomarkermolecule and a second type of binding ligand may be configured toassociate with the first binding ligand. In certain embodiments, forexample, both a first type of binding ligand and a second type ofbinding ligand may associate with the same or different epitopes of asingle biomarker molecule. In certain embodiments, for example, at leastone binding ligand may comprise an enzymatic component.

In certain embodiments, for example, a binding ligand and/or a biomarkermay comprise an enzymatic component. In certain embodiments, forexample, the enzymatic component may convert a precursor labeling agent(for example an enzymatic substrate) into a labeling agent (for examplea detectable product). In certain embodiments, for example, a measure ofthe concentration of biomarker molecules in the fluid sample may bedetermined based at least in part by determining the number of locationscontaining a labeling agent (for example by relating the number oflocations containing a labeling agent to the number of locationscontaining a biomarker molecule (or number of capture objects associatedwith at least one biomarker molecule to total number of captureobjects)). Non-limiting examples of enzymes or enzymatic componentsinclude horseradish peroxidase, beta-galactosidase, and alkalinephosphatase. Other non-limiting examples of systems or methods fordetection include embodiments where nucleic acid precursors arereplicated into multiple copies or converted to a nucleic acid that canbe detected readily, such as the polymerase chain reaction (PCR),rolling circle amplification (RCA), ligation, Loop-Mediated IsothermalAmplification (LAMP), etc. Such systems and methods will be known tothose of ordinary skill in the art, for example, as described in “DNAAmplification: Current Technologies and Applications,” Vadim Demidov etal., 2004.

In certain embodiments, for example, the biomarker molecules may beexposed to a precursor labeling agent (for example enzymatic substrate)and the enzymatic substrate may be converted to a detectable product(for example fluorescent molecule) upon exposure to a biomarkermolecule.

Certain embodiments may provide, for example, methods, tests, assays,kits, or systems that employ a variety of different components, steps,and/or other aspects that will be known and understood by those ofordinary skill in the art. In certain embodiments, for example, themethods, tests, assays, kits, or systems may comprise determining atleast one background signal determination. In certain embodiments, forexample, the methods, tests, assays, kits, or systems may furthercomprise subtracting the background signal from other determinations. Incertain embodiments, for example, the methods, tests, assays, kits, orsystems may comprise one more washings of capture objects to removeexcess sample, reagents, and the like.

In certain embodiments, for example, the methods, tests, assays, kits,or systems may include the use of at least one binding ligand. Incertain embodiments, for example, a measure of the concentration ofbiomarker molecules in a fluid sample may be based at least in part oncomparison of a measured parameter to a calibration curve. In certainembodiments, for example, the calibration curve may be formed at leastin part by determination at least one calibration factor.

In certain embodiments, for example, precursor labeling agents suspendedand/or solubilized in a liquid may be employed. In certain embodiments,for example, the precursor labeling agents may be converted to labelingagents which are insoluble in the liquid. In certain embodiments, forexample, the precursor labeling agents may be converted to labelingagents which become immobilized proximate a location (for example withinthe reaction vessel in which the labeling agent is formed). Suchprecursor labeling agents and labeling agents and their use aredescribed in commonly owned U.S. Patent Application Publication No.US-2010-0075862 (Ser. No. 12/236,484), filed Sep. 23, 2008, entitled“HIGH SENSITIVITY DETERMINATION OF THE CONCENTRATION OF ANALYTEMOLECULES OR PARTICLES IN A FLUID SAMPLE,” by Duffy et al., incorporatedherein by reference.

An exemplary embodiment of an assay method that may be used in certainembodiments of the invention is illustrated in FIG. 3A. A plurality ofcapture objects 2, are provided (step (A)). In this particular example,the plurality of capture objects comprises a plurality of beads. Thebeads are exposed to a fluid sample containing a plurality of biomarkermolecules 3 (for example beads 2 are incubated with biomarker molecules3). At least some of the biomarker molecules are immobilized withrespect to a bead. In this example, the biomarker molecules are providedin a manner (for example at a concentration) such that a statisticallysignificant fraction of the beads associate with a single biomarkermolecule and a statistically significant fraction of the beads do notassociate with any biomarker molecules. For example, as shown in step(B), biomarker molecule 4 is immobilized with respect to bead 5, therebyforming complex 6, whereas some beads 7 are not associated with anybiomarker molecules. It should be understood, in some embodiments, morethan one biomarker molecule may associate with at least some of thebeads, as described herein. At least some of the plurality of beads (forexample those associated with a single biomarker molecule or notassociated with any biomarker molecules) may then be spatiallyseparated/segregated into a plurality of locations. As shown in step(C), the plurality of locations is illustrated as substrate 8 comprisinga plurality of wells/reaction vessels 9. In this example, each reactionvessel comprises either zero or one beads. At least some of the reactionvessels may then be addressed (for example optically or via otherdetection means) to determine the number of locations containing abiomarker molecule. For example, as shown in step (D), the plurality ofreaction vessels are interrogated optically using light source 15,wherein each reaction vessel is exposed to electromagnetic radiation(represented by arrows 10) from light source 15. The light emitted(represented by arrows 11) from each reaction vessel is determined(and/or recorded) by detector 15 (in this example, housed in the samesystem as light source 15). The number of reaction vessels containing abiomarker molecule (for example reaction vessels 12) is determined basedon the light detected from the reaction vessels. In some cases, thenumber of reaction vessels containing a bead not associated with abiomarker molecule (for example reaction vessel 13), the number of wellsnot containing a bead (for example reaction vessel 14) and/or the totalnumber of wells addressed may also be determined. Such determination(s)may then be used to determine a measure of the concentration ofbiomarker molecules in the fluid sample.

A non-limiting example of an embodiment where a capture object isassociated with more than one biomarker molecule is illustrated in FIG.3B. A plurality of capture objects 20 are provided (step (A)). In thisexample, the plurality of capture objects comprises a plurality ofbeads. The plurality of beads is exposed to a fluid sample containingplurality of biomarker molecules 21 (for example beads 20 are incubatedwith biomarker molecules 21). At least some of the biomarker moleculesare immobilized with respect to a bead. For example, as shown in step(B), biomarker molecule 22 is immobilized with respect to bead 24,thereby forming complex 26. Also illustrated is complex 30 comprising abead immobilized with respect to three biomarker molecules and complex32 comprising a bead immobilized with respect to two biomarkermolecules. Additionally, in some cases, some of the beads may notassociate with any biomarker molecules (for example bead 28). Theplurality of beads from step (B) is exposed to a plurality of bindingligands 31. As shown in step (C), a binding ligand associates with someof the biomarker molecules immobilized with respect to a bead. Forexample, complex 40 comprises bead 34, biomarker molecule 36, andbinding ligand 38. The binding ligands are provided in a manner suchthat a statistically significant fraction of the beads comprising atleast one biomarker molecule become associated with at least one bindingligand (for example one, two, three, etc.) and a statisticallysignificant fraction of the beads comprising at least one biomarkermolecule do not become associated with any binding ligands. At least aportion of the plurality of beads from step (C) are then spatiallyseparated into a plurality of locations. As shown in step (D), in thisexample, the locations comprise a plurality of reaction vessels 41 on asubstrate 42. The plurality of reaction vessels may be exposed to theplurality of beads from step (C) such at each reaction vessel containszero or one beads. The substrate may then be analyzed to determine thenumber of reaction vessels containing a binding ligand (for examplereaction vessels 43), wherein in the number may be related to a measureof the concentration of biomarker molecules in the fluid sample. In somecases, the number of reaction vessels containing a bead and notcontaining a binding ligand (for example reaction vessel 44), the numberof reaction vessels not containing a bead (for example reaction vessel45), and/or the total number of reaction vessels addressed/analyzed mayalso be determined. Such determination(s) may then be used to determinea measure of the concentration of biomarker molecules in the fluidsample.

In certain embodiments, for example, a plurality of locations may beaddressed and/or a plurality of capture objects and/orspecies/molecules/particles of interest may be detected substantiallysimultaneously. “Substantially simultaneously” when used in thiscontext, refers to addressing/detection of the locations/captureobjects/species/molecules/particles of interest at approximately thesame time such that the time periods during which at least twolocations/capture objects/species/molecules/particles of interest areaddressed/detected overlap, as opposed to being sequentiallyaddressed/detected, where they would not. Simultaneousaddressing/detection may be accomplished by using various techniques,including optical techniques (for example CCD detector). In certainembodiments, for example, capture objects/species/molecules/particlesmay be spatially segregated into a plurality of discrete, resolvablelocations. In certain embodiments, for example, the spatially segregatedcapture objects/species/molecules/particles may be detectedsubstantially simultaneously by allowing multiple locations to beaddressed substantially simultaneously. In certain embodiments, forexample, individual species/molecules/particles may be associated withcapture objects that are spatially segregated with respect to the othercapture objects into a plurality of discrete, separately resolvablelocations during detection. In certain embodiments, for example, theplurality of discrete, individual capture objects (and thus individualspecies/molecules/particles (for example biomarker molecules)) areseparately resolved in the separately resolvable locations. In certainembodiments, for example, individual molecules/particles of a pluralityof molecules/particles may be partitioned across a plurality of reactionvessels such that each reaction vessel contains zero or only onespecies/molecule/particle. In certain embodiments, for example, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 96%, at least about 97%, at least about 98%, at leastabout 99%, at least about 99.5% of all species/molecules/particles maybe spatially separated with respect to other species/molecules/particlesduring detection. In certain embodiments, for example, a plurality ofspecies/molecules/particles may be detected substantially simultaneouslywithin a time period of less than about 1 second, less than about 500milliseconds, less than about 100 milliseconds, less than about 50milliseconds, less than about 10 milliseconds, less than about 1millisecond, less than about 500 microseconds, less than about 100microseconds, less than about 50 microseconds, less than about 10microseconds, less than about 1 microsecond, less than about 0.5microseconds, less than about 0.1 microseconds, or less than about 0.01microseconds, less than about 0.001 microseconds, or less. In certainembodiments, for example, the plurality of species/molecules/particlesmay be detected substantially simultaneously within a time period ofbetween about 100 microseconds and about 0.001 microseconds, betweenabout 10 microseconds and about 0.01 microseconds, or less.

In certain embodiments, for example, the locations may be opticallyinterrogated. In certain embodiments, for example, locations exhibitingchanges in their optical signature may be identified by a conventionaloptical train and optical detection system. Depending on the detectedspecies (for example type of fluorescence entity, etc.) and theoperative wavelengths, optical filters designed for a particularwavelength may be employed for optical interrogation of the locations.In certain embodiments, for example, a system for optical interrogationmay comprise more than one light source and/or a plurality of filters toadjust the wavelength and/or intensity of the more than one lightsource. In certain embodiments, for example, the optical signal from aplurality of locations may be captured using a CCD camera.

In certain embodiments, for example, the plurality of reaction vesselsmay be sealed (for example after the introduction of the biomarkermolecules, binding ligands, and/or precursor labeling agent). In certainembodiments, for example, the plurality of reaction vessels may besealed by mating of the second substrate with a sealing component. Incertain embodiments, for example, the plurality of reaction vessels maybe sealed with a sealing fluid (for example a silicone oil sealingfluid). In certain embodiments, for example, the sealing of the reactionvessels may be such that the contents of each reaction vessel cannotescape the reaction vessels during performance of an assay. In certainembodiments, for example, the reaction vessels may be sealed after theaddition of the biomarker molecules and, optionally, at least one typeof precursor labeling agent to facilitate detection of the biomarkermolecules. In certain embodiments, for example, by sealing the contentsin some or each reaction vessel, a reaction to produce the detectablelabeling agents may proceed within the sealed reaction vessels, therebyproducing a detectable amount of labeling agents that is retained in thereaction vessel for detection purposes.

In certain embodiments, for example, the plurality of locations may beformed using a variety of methods and/or materials. In certainembodiments, for example, the plurality of locations may comprise aplurality of reaction vessels/wells on a substrate. In certainembodiments, for example, the plurality of reaction vessel may be formedas an array of depressions on a first surface. In certain embodiments,for example, the plurality of reaction vessels may be formed by mating asealing component comprising a plurality of depressions with a substratethat may either have a featureless surface or include depressionsaligned with those on the sealing component. Any of the devicecomponents, for example, the substrate or sealing component, may befabricated from a compliant material, for example an elastomeric polymermaterial, to aid in sealing. The surfaces may be or made to behydrophobic or contain hydrophobic regions to minimize leakage ofaqueous samples from the microwells. In certain embodiments, forexample, the reactions vessels may be configured to receive and containonly a single capture object (for example a single bead such as aparamagnetic bead).

In certain embodiments, for example, the reaction vessels may all haveapproximately the same volume. In certain embodiments, for example, thereaction vessels may have differing volumes. In certain embodiments, forexample, the volume of each individual reaction vessel may be selectedto be appropriate to facilitate an assay protocol. In certainembodiments, for example, the number of capture objects used forbiomarker capture contained in each vessel may be limited to a smallnumber. In certain further embodiments, for example, the volumes of thereaction vessels may range from 1 attoliter (or smaller) to 100nanoliters (or larger). In certain further embodiments, for example, thevolumes of the reaction vessels may be selected depending upon one ormore of the nature of the capture objects, the detection technique andequipment employed, the number and density of the wells on thesubstrate, and the expected concentration of capture objects in thefluid applied to the substrate containing the wells. In certainembodiments, for example, the size of one or more of the reactionvessels may be selected such only a single capture object used forbiomarker capture can be fully contained within the reaction vessel. Incertain embodiments, for example, the size of one or more of thereaction vessels may be selected according to one or more of theembodiments disclosed in U.S. patent application Ser. No. 12/731,130,filed Mar. 24, 2010, published as US-2011-0212848 on Sep. 1, 2011,entitled “ULTRA-SENSITIVE DETECTION OF MOLECULES OR PARTICLES USINGBEADS OR OTHER CAPTURE OBJECTS,” by Duffy et al.; or InternationalPatent Application No. PCT/US2011/026645, filed Mar. 1, 2011, publishedas WO 2011/109364 on Sep. 9, 2011, entitled “ULTRA-SENSITIVE DETECTIONOF MOLECULES OR PARTICLES USING BEADS OR OTHER CAPTURE OBJECTS,” byDuffy et al., each herein incorporated by reference.

In certain embodiments, for example, the reaction vessels may have avolume between about 1 femtoliter and about 1 picoliter, between about 1femtoliters and about 100 femtoliters, between about 10 attoliters andabout 100 picoliters, between about 1 picoliter and about 100picoliters, between about 1 femtoliter and about 1 picoliter, or betweenabout 30 femtoliters and about 60 femtoliters. In certain embodiments,for example, the reaction vessels have a volume of less than about 1picoliter, less than about 500 femtoliters, less than about 100femtoliters, less than about 50 femtoliters, or less than about 1femtoliter. In certain embodiments, for example, the reaction vesselshave a volume of about 10 femtoliters, about 20 femtoliters, about 30femtoliters, about 40 femtoliters, about 50 femtoliters, about 60femtoliters, about 70 femtoliters, about 80 femtoliters, about 90femtoliters, or about 100 femtoliters.

In certain embodiments, for example, the total number of locationsand/or density of the locations employed in an assay. In certainembodiments, for example, the total number of locations and/or densityof the locations of reaction vessels in an array of reaction vessels candepend on the composition and end use of the array. In certainembodiments, for example, the number of reaction vessels employed maydepend on the number of types of biomarker molecule and/or bindingligand employed, the suspected concentration range of the assay, themethod of detection, the size of the capture objects, the type ofdetection entity (for example free labeling agent in solution,precipitating labeling agent, etc.). In certain embodiments, forexample, arrays containing from about 2 to more than 1 billion reactionvessels (or total number of reaction vessels) may be made by utilizingany one of a variety of techniques and materials. In certainembodiments, for example, increasing the number of reaction vessels inthe array may be used to increase the dynamic range of an assay or toallow multiple samples or multiple types of biomarker molecules to beassayed in parallel. In certain embodiments, for example, the array maycomprise between one thousand and one million reaction vessels persample to be analyzed. In certain embodiments, for example, the arraymay comprise greater than one million reaction vessels. In certainembodiments, for example, the array may comprise between about 1,000 andabout 50,000, between about 1,000 and about 1,000,000, between about1,000 and about 10,000, between about 10,000 and about 100,000, betweenabout 100,000 and about 1,000,000, between about 100,000 and about500,000, between about 1,000 and about 100,000, between about 50,000 andabout 100,000, between about 20,000 and about 80,000, between about30,000 and about 70,000, between about 40,000 and about 60,000 reactionvessels. In certain embodiments, for example, the array may compriseabout 10,000, about 20,000, about 50,000, about 100,000, about 150,000,about 200,000, about 300,000, about 500,000, about 1,000,000, or more,reaction vessels.

In certain embodiments, for example, the array of reaction vessels maybe arranged on a substantially planar surface or in a non-planarthree-dimensional arrangement. In certain embodiments, for example, thereaction vessels may be arrayed in a regular pattern or may be randomlydistributed. In certain embodiments, for example, the array may be aregular pattern of sites on a substantially planar surface permittingthe sites to be addressed in an X-Y coordinate plane.

In certain embodiments, for example, the reaction vessels may be formedin a solid material. Suitable materials in which the reaction vesselscan be formed includes, but is not limited to, glass (including modifiedand/or functionalized glass), plastics (including acrylics, polystyreneand copolymers of styrene and other materials, polypropylene,polyethylene, polybutylene, polyurethanes, cyclic olefin copolymer(COC), cyclic olefin polymer (COP), Teflon®, polysaccharides, nylon ornitrocellulose, etc.), elastomers (such as poly(dimethyl siloxane) andpoly urethanes), composite materials, ceramics, silica or silica-basedmaterials (including silicon and modified silicon), carbon, metals,optical fiber bundles, or the like. In certain embodiments, for example,the substrate material may be selected to allow for optical detectionwithout appreciable autofluorescence. In certain embodiments, forexample, the reaction vessels may be formed in a flexible material.

A reaction vessel in a surface (for example a substrate or a sealingcomponent) may be formed using a variety of techniques known in the art,including, but not limited to, photolithography, stamping techniques,molding techniques, etching techniques, or the like. As will beappreciated by those of the ordinary skill in the art, the techniqueused can depend on the composition and shape of the supporting materialand the size and number of reaction vessels. In a particular embodiment,an array of reaction vessels is formed by creating microwells on one endof a fiber optic bundle and utilizing a planar compliant surface as asealing component.

In certain embodiments, for example, the methods and assays may becarried out using commercially available systems, for example, the SimoaHD-1 Analyzer and Quanterix SR-X (Quanterix™, Lexington, Mass.) and thesystems described in U.S. patent application Ser. No. 13/035,472, filedFeb. 25, 2011, published as US 2012-0196774, entitled “SYSTEMS, DEVICES,AND METHODS FOR ULTRA-SENSITIVE DETECTION OF MOLECULES OR PARTICLES,” byFournier et al., herein incorporated by reference.

In certain embodiments, for example, the array of reaction vessels maybe fabricated using other methods and materials that do not utilize theends of an optical fiber bundle as a substrate. In certain embodiments,for example, the array may be a spotted, printed orphotolithographically fabricated substrate produced by techniques knownin the art such as those disclosed in WO95/25116; WO95/35505; PCTUS98/09163; U.S. Pat. Nos. 5,700,637, 5,807,522, 5,445,934, 6,406,845,and 6,482,593, which are hereby incorporated, in their entirety, byreference. In certain embodiments, for example, the array may beproduced using molding, embossing, and/or etching techniques as will beknown to those of ordinary skill in the art.

In certain embodiments, for example, the plurality of locations may notcomprise a plurality of reaction vessels/wells. In certain embodiments,for example, a patterned substantially planar surface may be employedand the patterned areas form a plurality of locations for receiving thecapture objects. In certain embodiments, for example, the patternedareas may comprise substantially hydrophilic surfaces which aresubstantially surrounded by substantially hydrophobic surfaces. Incertain embodiments, for example, a plurality of capture objects (forexample beads) may be substantially surrounded by a substantiallyhydrophilic medium (for example comprising water), and the beads may beexposed to the patterned surface such that the beads associate in thepatterned areas (for example the hydrophilic locations on the surface),thereby spatially segregating the plurality of beads. In certainembodiments, for example, a substrate may be or include a gel or othermaterial able to provide a sufficient barrier to mass transport (forexample convective and/or diffusional barrier) to prevent captureobjects used for biomarker capture and/or precursor labeling agentand/or labeling agent from moving from one location on or in thematerial to another location so as to cause interference or cross-talkbetween spatial locations containing different capture objects duringthe time frame required to address the locations and complete the assay.In certain embodiments, for example, a plurality of capture objects maybe spatially separated by dispersing the capture objects on and/or in ahydrogel material. In certain embodiments, for example, a precursorlabeling agent may be already present in the hydrogel, therebyfacilitating development of a local concentration of the labeling agent(for example upon exposure to a binding ligand or biomarker moleculecarrying an enzymatic component). In certain embodiments, for example,the capture objects may be confined in one or more capillaries. Incertain embodiments, for example, the plurality of capture objects maybe absorbed or localized on a porous or fibrous substrate (for examplefilter paper). In certain embodiments, for example, the capture objectsmay be spatially segregated on a uniform surface (for example a planarsurface), and the capture objects may be detected using precursorlabeling agents which are converted to substantially insoluble orprecipitating labeling agents that remain localized at or near thelocation of where the corresponding capture object is localized. The useof such substantially insoluble or precipitating labeling agents isdescribed herein. In certain embodiments, for example, single biomarkermolecules may be spatially segregated into a plurality of droplets. Incertain embodiments, for example, single biomarker molecules may besubstantially contained in a droplet containing a first fluid. Incertain embodiments, for example, the droplet may be substantiallysurrounded by a second fluid, wherein the second fluid is substantiallyimmiscible with the first fluid.

Certain embodiments may provide, for example, methods and assayscomprising at least one washing. In certain embodiments, for example, awash solution for the washing is selected so that it does not causeappreciable change to the configuration of the capture objects. Incertain embodiments, for example, a wash solution for the washing isselected so that it does not cause appreciable change to the biomarkermolecules. In certain embodiments, for example, a wash solution for thewashing is selected so that it does not disrupt any specific bindinginteraction between at least two components (for example between acapture component and a biomarker molecule component). In certainembodiments, for example, the wash solution may be a solution that isselected to chemically interact with one or more assay components. Aswill be understood by those of ordinary skill in the art, a wash stepmay be performed at any appropriate time point during the methods andassays. In certain embodiments, for example, a plurality of captureobjects may be washed after exposing the capture objects to one or moresolutions comprising biomarker molecules, binding ligands, precursorlabeling agents, or the like. In certain embodiments, for example,following immobilization of the biomarker molecules with respect to aplurality of capture objects, the plurality of capture objects may besubjected to a washing step thereby removing any biomarker molecules notspecifically immobilized with respect to a capture object.

Other assay methods in addition to those described herein are known inthe art and may be used in connection with the inventive methods. Incertain embodiments, for example, various analyzers are commerciallyavailable for the determination of the concentration of biomarkers. Theassay methods employed should meet the algorithm requirements for LODand LOQ.

INCORPORATION BY REFERENCE

Without limitation, the following documents are hereby incorporated, intheir entirety, by reference: U.S. Patent Application Publication Nos.2002/0122612, 2003/0016897, 2003/0027126, 2005/0130188, 2006/0013543,2007/0040095, 2007/0122861, 2007/0259448, 2007/0259385, 2007/0259381,2008/0032324, 2009/0101175, 2009/0149341, 2009/0156425, 2009/0170728,2010/0075862, 2010/0075407, 2010/0075439, 2010/0075355, 2011/0195852,2011/0212848, 2011/0212462, 2011/0212537, 2011/0245097, 2011/0251105,2012/0135154, 2012/0196774, 2012/0277114, 2013/0034284, 2013/0142710,2013/0266969, 2014/0094386, 2014/0134652, 2014/0227720, 2014/0302532,2015/0038355, 2015/0353997, 2016/0123969, 2017/0160292, 2018/0003703,2018/0037614, and 2018/0339296; U.S. Pat. No. 6,706,526; U.S.Provisional Application Nos. 62/655,738 and 62/789,067; InternationalPatent Publication Nos. WO 2009/029073, WO 2010/039179, WO 2011/109364,WO 2011/109372, WO 2011/109379, WO 2016/115256, WO 2016/130923, andWO2018/222585; Kuhle, J. et al., “Comparison of three analyticalplatforms for quantification of the neurofilament light chain in bloodsamples: ELISA, electrochemiluminescence immunoassay and Simoa,” ClinChem Lab Med 54:10 (2016) 1655-1661; Rissin, D. M. et al., “SimultaneousDetection of Single Molecules and Singulated Ensembles of MoleculesEnables Immunoassays with Broad Dynamic Range,” Anal Chem 83:6 (2011)2279-2285; Armbruster, D. A. et al., “Limit of Blank, Limit of Detectionand Limit of Quantitation,” Clin Biochem Rev 29(Suppl 1) (2008) S49-S52;Quanterix, “Neuro 4-Plex A Kit: Material Safety Data Sheet,” (May 25,2017), available athttps://www.quanterix.com/sites/default/files/safety_data_sheets/Neuro%204-Plex%20A%20Kit%20102153.pdf;Harlow and Lane, (1988) Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, New York; Demidov, V. et al., “DNA Amplification:Current Technologies and Applications,” J Antimicrobial Chemotherapy54:6 (2004); Depoorter et al., “Neurofilament Light Chain: BloodBiomarker of Neonatal Neuronal Injury” Front. Neurol. (20 Nov. 2018),available athttps://www.frontiersin.org/articles/10.3389/fneur.2018.00984/full;Gill, J. et al., “Glial fibrillary acidic protein elevations relate toneuroimaging abnormalities after mild TBI,” Neurology 91:15 (Oct. 9,2018) e1385-e1389; Korley, F. K., “Performance Evaluation of a MultiplexAssay for Simultaneous Detection of Four Clinically Relevant TraumaticBrain Injury Biomarkers,” J. Neurotrauma 36 (Jan. 1, 2019) 182-187;Hossain, I. et al., “Early Levels of Glial Fibrillary Acidic Protein andNeurofilament Light Protein in Predicting the Outcome of Mild TraumaticBrain Injury,” J. Neurotrauma 36 (2019) 1-10; Good, N. E. et al.,“Hydrogen Ion Buffers for Biological Research,” Biochemistry 5:2 (1966)467-477; Crowther, J R., “ELISA: Theory and Practice Methods,” Mol.Biol. 42 (1995) 1-218; Davies, C., “Concepts,” In the ImmunoassayHandbook, D. Wild, ed., Stockton Press, New York (1994) 83-115;Hornbeck, P., “Enzyme-Linked Immunosorbent Assays,” In Current Protocolsin Immunology, Unit 2.1, ed., R. Coico, John Wiley & Sons, Hoboken, N.J.(2001); and Pirkanniemi, K. “Complexing Agents Long-term Toxicity,Catalytic Oxidative Degradation and Concentrations in Industrial WasteWater,” Doctoral Dissertation, University of Kuopio (2007)(collectively, the “INCORPORATED REFERENCES”).

Examples

In the following Examples:

“AEB” means “average enzyme per bead”;

“>” means “greater than”;

“<” means “less than”;

“LOB” means “limit of blank”;

“LOD” means “limit of detection”;

“LOQ” means “limit of quantification”;

“q.s.” refers to a quantity of buffer sufficient to bring the listedcomponents to the concentrations indicated;

“n.a.” means “not applicable”; and

“---” indicates no data presented.

“no.” means “number”;

“SD” means “standard deviation”;

“NSI” means “Neurobehavioral Symptom Inventory”;

“MVA” means “motor vehicle accident”;

“+” means positive indication of brain injury;

“−” means negative indication of brain injury;

“S” means “statistically significant difference”; and

“NS” means “nonsignificant difference”.

Example 1. The following non-limiting example describes a humanmultiplex assay that measured four important neurology biomarkers inboth cerebrospinal fluid (CSF) and blood. The four targets were NF-L,total tau protein, glial GFAP, and UCH L1. All four biomarkers have beenstudied as indicators of TBI severity. Recent reports indicate serumNF-L is a biomarker for mild TBI in amateur boxers and professionalhockey players1, that plasma tau is related to concussion severity, andthat serum GFAP and UCH L1 can detect mild to moderate TBI. The fourtargets were measured in a single performance of the assay. Each assayof the multiplex assay is described below.

NF-L is a 68 kDa cytoskeletal intermediate filament protein that isexpressed in neurons. It associates with the 125 kDa Neurofilamentmedium (NF-M) and the 200 kDa Neurofilament heavy (NF-H) to formneurofilaments. They are major components of the neuronal cytoskeleton,and are believed to function primarily to provide structural support forthe axon and to regulate axon diameter. Neurofilaments can be releasedin significant quantity following axonal damage or neuronaldegeneration. NF-L has been shown to associate with TBI, MS,frontotemporal dementia and other neurodegenerative diseases. The NF-Lassay was a digital immunoassay for the quantitative determination ofNF-L in serum, plasma and CSF. The antibodies also cross reacted withmurine, bovine, and macaque NF-L epitopes, and the assay could be usedfor research with these species.

FIG. 4A shows the NF-L immunoassay dose-response curve. Calibrators wereassayed in replicates of three. Four-parameter curve fit parameters aredepicted. The analytical performance characteristics of the NF-L portionof the multiplex assay were characterized as summarized in Table 1below. Table 1 shows the reproducibility of the NF-L immunoassay portionof the multiplex assay. FIG. 4B shows the NF-L concentration in EDTAplasma (n=20), matched serum (n=20) and CSF (n=20) from healthy donors.Error bars depict mean and interquartile ranges.

TABLE 2 Reproducibility precision of NF-L immunoassay across instrumentsand reagent lots. Reproducibility was determined with guidance from CLSIProtocol EP5-A. Five samples consisting of two serum panels, one plasmapanel, and two NF-L controls were assayed in replicates of three for tworuns on each of three instruments and two reagent lots. Analysis ofvariance (nested ANOVA) results are summarized in the following table.Between Between Between Within Mean Instrument Lot Run Run NF-L % CV, %CV, % CV, % CV, Sample pg/mL n = 3 n = 2 n = 12 n = 30 Control 1 9.310.9 3.4 5.8 6.2 Control 2 925 0.0 6.1 4.6 4.4 Serum Panel 1 16.8 0.0 0.05.1 5.4 Plasma Panel 2 60.1 0.0 0.0 5.6 4.7 Serum Panel 3 332 0.0 3.75.0 4.7

Tau is a microtubule-stabilizing protein primarily localized in centralnervous system neurons, but also expressed at low levels in astrocytesand oligodendrocytes. Tau consists of six isoforms in the human brain,with molecular weights of 48,000 to 67,000 Daltons depending on isoform.Tau elevation is observed in the cerebrospinal fluid (CSF) of patientswith neurodegenerative disease and head injuries, suggesting itsextracellular release during neuronal damage and a role as a biomarkerwith specificity for brain injury. Potential movement of elevated CSFtau protein across the blood-brain barrier presents a possibility thatmeasurements of tau protein in blood could provide a convenientperipheral window into brain/CSF status. Studies of tau protein in serumand plasma have been hampered by its low abundance (typically lowpg/mL), and there are relatively few reports characterizing theappearance of tau protein in blood or evaluating the usefulness of thisbiomarker for brain injury assessment. Recent reports using digitalimmunoassay technology have shown elevation in peripheral tau proteinassociated with hypoxic brain injury, concussed hockey players, andrepetitive minimal head injury in Olympic boxing. The total tau portionof the multiplex assay used a combination of monoclonal antibodies thatreact with both normal and phosphorylated tau. With an epitope in themidregion of the molecule, the assay recognizes all tau isoforms.

FIG. 5A shows the tau immunoassay dose-response curve. Calibrators wereassayed in replicates of three. Four-parameter curve fit parameters aredepicted. The analytical performance characteristics of the tau portionof the multiplex assay were characterized as summarized in Table 3below. Table 4 shows the reproducibility of the NF-L immunoassay portionof the multiplex assay. FIG. 5B shows the tau concentration in EDTAplasma (n=20), matched serum (n=20) and CSF (n=20) from healthy donors.Error bars depict mean and interquartile ranges.

TABLE 4 Reproducibility precision of tau immunoassay across instrumentsand reagent lots. Reproducibility was determined with guidance from CLSIProtocol EP5-A. Five samples consisting of two serum panels, one plasmapanel, and two tau controls were assayed in replicates of three for tworuns on each of three instruments and two reagent lots. Analysis ofvariance (nested ANOVA) results are summarized in the following table.Between Between Between Within Mean Instrument Lot Run Run Tau % CV, %CV, % CV, % CV, Sample pg/mL n = 3 n = 2 n = 12 n = 30 Control 1 3.717.1 2.6 1.6 7.6 Control 2 200 0.0 6.7 4.5 4.5 Serum Panel 1 2.66 0.0 0.05.7 6.7 Plasma Panel 2 20.2 1.5 0.0 6.6 4.9 Serum Panel 3 148 5.2 0.03.0 5.2

GFAP is a class-III intermediate filament majorly expressed inastrocytic glial cells in the central nervous system. Astrocytes play avariety of key roles in supporting, guiding, nurturing, and signalingneuronal architecture and activity. Monomeric GFAP is about 55 kD. It iscapable of forming both homodimers and heterodimers; GFAP can polymerizewith other type III proteins or with neurofilament protein, such asNF-L. GFAP is involved in many important CNS processes, including cellcommunication and the functioning of the blood brain barrier. GFAP, as apotential biomarker has been shown to associate with multiple diseasessuch as TBI, stroke, brain tumors, etc. Decreases in GFAP expressionhave been reported in Down's syndrome, schizophrenia, bipolar disorder,and depression.

FIG. 6A shows the tau immunoassay dose-response curve. Calibrators wereassayed in replicates of three. Four-parameter curve fit parameters aredepicted. The analytical performance characteristics of the GFAP portionof the multiplex assay were characterized as summarized in Table 5below. Table 6 shows the reproducibility of the GFAP immunoassay portionof the multiplex assay. FIG. 6B shows the GFAP concentration in EDTAplasma (n=20), matched serum (n=20) and CSF (n=20) from healthy donors.Error bars depict mean and interquartile ranges.

TABLE 6 Reproducibility precision of GFAP immunoassay across instrumentsand reagent lots. Reproducibility was determined with guidance from CLSIProtocol EPS-A. Five samples consisting of two serum panels, one plasmapanel, and two GFAP controls were assayed in replicates of three for tworuns on each of three instruments and two reagent lots. Analysis ofvariance (nested ANOVA) results are summarized in the following table.Between Between Between Within Mean Instrument Lot Run Run GFAP % CV, %CV, % CV, % CV, Sample pg/mL n = 3 n = 2 n = 12 n = 30 Control 1 27.60.0 3.9 3.0 7.7 Control 2 1492 0.0 2.2 7.1 4.2 Serum Panel 1 59.4 0.08.5 3.8 3.9 Plasma Panel 2 226 0.0 3.0 6.2 3.6 Serum Panel 3 2095 0.06.5 3.8 4.5

The UCH L1, hydrolyze small C-terminal adducts of ubiquitin to generatethe ubiquitin monomer. It is also called PARK5 or neuronal-specificprotein gene product 9.5, expressed predominantly in neurons, UCH L1 isone of the most abundant brain protein, representing 1 to 2% of totalsoluble brain protein. In vivo, UCH L1 has been shown to be involved inthe regulation of the ubiquitin pool, apoptosis, learning and memory,and its absence in mice because of spontaneous intragenic deletionsyields phenotypes with neurological defects. A point mutation (193M) anda polymorphism (S18Y) in this gene have been shown to associate withParkinson's disease. Recently, UCH L1 has been proposed as a candidatebiomarker for brain injury. UCH L1 can be released from injured neuronsand flow into the cerebrospinal fluid and circulating blood.

FIG. 7A shows the tau immunoassay dose-response curve. Calibrators wereassayed in replicates of three. Four-parameter curve fit parameters aredepicted. The analytical performance characteristics of the UCH L1portion of the multiplex assay were characterized as summarized in Table7 below. Table 8 shows the reproducibility of the UCH L1 immunoassayportion of the multiplex assay. FIG. 7B shows the UCH L1 concentrationin EDTA plasma (n=20), matched serum (n=20) and CSF (n=20) from healthydonors. Error bars depict mean and interquartile ranges.

TABLE 8 Reproducibility precision of UCH L1 immunoassay acrossinstruments and reagent lots. Reproducibility was determined withguidance from CLSI Protocol EP5-A. Five samples consisting of two serumpanels, one plasma panel, and two UCH-L1 controls were assayed inreplicates of three for two runs on each of three instruments and tworeagent lots. Analysis of variance (nested ANOVA) results are summarizedin the following table. Between Between Between Within Mean InstrumentLot Run Run UCH-L1 % CV, % CV, % CV, % CV, Sample pg/mL n = 3 n = 2 n =12 n = 30 Control 1 129 1.8 0.0 2.9 9.3 Control 2 13145 0.0 2.8 4.9 5.4Serum Panel 1 71.9 5.0 0.0 6.4 11.3 Plasma Panel 2 381 4.8 0.0 4.9 5.6Serum Panel 3 6663 0.0 4.2 5.9 4.2

Example 3. This example describes the materials and methods used inExample 3.

Instrumentation

The multiplex assay in Example 3 was formulated for use on eitherQuanterix Simoa HD-1 Analyzer or Simoa SR-X (Quanterix™, Lexington,Mass.). The Simoa HD-1 is a fully automated digital immunoassay analyzerthat utilizes single molecule array (Simoa) technology for isolation andcounting of single molecules. The instrument pipettes sample directlyfrom sample tubes (capacity 96 tubes) and processes immunoassays anddata reduction with a steady state throughput of 66 tests/hour. TheSimoa SR-X is a semi-automated digital immunoassay detection system thatfunctions like the HD-1 system except that samples and reagents areadded manually to a 96-well ELISA plate for the assay-processing step.Following completion of the immunoassay processing, the ELISA plates areimaged automatically by the plate loading system, and processed insimilar fashion to the HD-1. The multiplex assay in Example 3 was run onSimoa SR-X.

Reagents

The following four reagents were developed: paramagnetic capture beads,biotinylated detector molecules, streptavidin: β-galactosidase (SβG)conjugate, and sample diluent. The capture beads comprised a mixture ofparamagnetic microbeads conjugated separately with anti-NF-L mousemonoclonal antibodies, anti-tau mouse monoclonal antibodies, anti-GFAPmouse monoclonal antibodies, and anti-UCH L1 mouse monoclonalantibodies. For multiplexing, the beads were pre-coated with fourseparate fluorescent dyes, one dye for each analyte. Each fluorescentlylabeled bead type was assigned to one particular analyte (488 nm dye toNF-L, 750 nm dye to tau, 647 nm dye GFAP, and 700 nm dye to UCH L1. Thebeads were not co-coated with all four antibodies, but were coatedseparately in independent batches, then mixed together in equalportions. The antibodies were covalently attached to the beads bystandard carbodiimide coupling chemistry to 2.7-μm carboxy paramagneticmicrobeads (Agilent Technologies). The antibody-coated beads werediluted to a concentration of 7×10⁶ beads/mL in a diluent composed of 50mM Tris (pH 7.8), 50 mM NaCl, 10 mM EDTA, 1% BSA, 0.1% Tween 20, and anantimicrobial preservative (for example ProClin™ 300). Biotinylateddetector reagent comprised a mixture of four biotinylated mousemonoclonal antibodies directed to epitopes on NF-L, tau, GFAP, and UCHL1. The antibodies were separately biotinylated using standard methodsknown in the art (for example EZ-Link NHS-PEG4-Biotin, LifeTechnologies), prior to mixing. Antibodies were diluted in such a way asto maximize signal while minimizing non-specific binding. Detectorconcentrations for the 4-plex differed significantly from what wasutilized for single assays, and the range of detector concentrations was0.1 mcg/mL (NF-L) to 1.8 mcg/mL (tau). Dilutions were performed in adiluent of phosphate-buffered saline (pH 7.4), 2% BSA, 0.1% Tween 20 andan anti-microbial preservative. SβG reagent was prepared by covalentconjugation of purified streptavidin (Thermo Scientific) and RG (Sigma)using standard coupling chemistry and diluted to 150 pM in phosphatebuffered saline (pH 7.4) with 1% sucrose, 3% BSA, 0.1% Tween 20, and 1mM MgCl₂ with an anti-microbial preservative. Sample diluent wasformulated in phosphate buffered saline (pH 7.4) with dextrose (0.06%),BSA (0.02%), BgG (0.01%), urea (5 mM), Triton™ X-100 (0.5%), TRU Block™(10 mcg/mL), Superchemiblock™ (0.05%), and an anti-microbialpreservative. This unique and complex formulation resulted in goodanalytical characteristics for the multiplex assay.

Calibration

Multi-constituent calibrators were prepared in a diluent of phosphatebuffered saline, pH 7.4, with 2.7 mM KCl, 2% BSA, 0.1% 10G Surfactant,10 mcg/mL TRU Block™, 5 mM EDTA, and of an anti-microbial (ProClin™300). The following antigens used were:

NF-light: Encor, recombinant human full length NF-L, Vendor Part#Prot-r-NF-L

Tau: Millipore Tau 381 (AG952)

GFAP: Hytest, native human GFAP, Vendor Part #8GF23

UCH L1: Origene, recombinant human UCHL1, Vendor Part #TP301803. Asdescribed in the next section, the choice of GFAP antigen was importantin reducing cross reactivity with the other three assays. Calibratorlevels were:

NF-light: 0, 0.5, 1.5, 5, 15, 50, 150, 500 pg/mL,

Tau: 0, 0.1, 0.3, 1, 3, 10, 30, 100 pg/mL;

GFAP: 0, 1, 3, 10, 30, 100, 300, 1000 pg/mL;

UCH L1: 0. 10, 30, 100, 300, 1000, 3000, 10000 pg/mL.

Protocol

The multiplex assay in Example 3 was a 2-step digital immunoassay. Inthe first step, assay specific antibody coated paramagnetic capturebeads, sample, and biotinylated detector antibody were combined. Thesebeads were labeled with different dyes. NF-L, tau, GFAP, and UCH L1molecules present in the sample were captured by the correspondentantibody coated capture beads and labeled with biotinylated detectorantibodies. After washing, a conjugate of streptavidin-ß-galactosidase(SBG) is mixed with the capture beads. SBG binds to the biotinylateddetector antibodies, resulting in enzyme labeling of captured targets.Following a second wash, the capture beads are resuspended in aresorufin ß-D-galactopyranoside (RGP) substrate solution and transferredto the Simoa Disc. Individual capture beads are then sealed withinmicrowells in the array. If target has been captured and labeled, theß-galactosidase hydrolyzes the RGP substrate into a fluorescent productthat provides the signal for measurement. A single labeled targetmolecule results in sufficient fluorescent signal to be detected andcounted by the Simoa optical system. The dye signal on each bead isdecoded to determine which target it captured. At low targetconcentration, the percentage of bead-containing wells in the array thathave a positive signal is proportional to the amount of target presentin the sample. At higher target concentration, when most of thebead-containing wells have one or more labeled target molecules, thetotal fluorescence signal is proportional to the amount of targetpresent in the sample. The concentration of NF-L, Tau, GFAP, and UCH L1in unknown samples is interpolated from a standard curve.

When running the described reagents for the 4-plex on the HD-1, theabove protocol was executed automatically by the instrument. Whenutilizing the SR-X instrument, the above protocol was performed outsideof the instrument with manual pipetting by the user. The followingsequence of steps were used to perform the testing manually inpreparation for detection with the SR-X system. First, sample dilutionswere prepared were diluted 4× in sample diluent. Then, aliquots ofdiluted samples were pipetted into ELISA plate wells. Third, capturebeads were pipetted into each ELISA plate well (20 μl per well). Next,detector molecules were pipetted into each ELISA well (20 μl per well).Then, the sample plate was incubated on a microplate shaker for 30minutes, 800 rpm, 30° C. Next, the sample plate was washed using amicroplate washer (for example BioTek washer) with 3 cycles with Triswash buffer. The total wash time was 5 minutes. Then SBG reagent wasadded (100 μl per well). Next, a second wash was performed using amicroplate washer (for example BioTek washer) with 2 cycles withsucrose-containing wash buffer. The total wash time was 2 minutes. Then,the plate was incubated on magnet for 10 minutes to dry pellet. Once,the assay prep was complete, the plate was transferred to SR-X and startrun.

Example 4. The following non-limiting example describes the non-specificbinding of certain GFAP antigen used for calibration. Table A shows theaverage enzyme per bead (AEB) and the S/B for a calibration curve usingGFAP Vendor An antigen and GFAP Hytest antigen.

TABLE A Antigen Beads Tau Ab42 GFAP UCHL1 Detector Det Mix Det Mix DetMix Det Mix Calibrator pg/mL AEB S/B AEB S/B AEB S/B AEB S/B GFAP VendorA Cal A 0 0.035 0.020 0.012 0.007 Cal B 0.122 0.033 1.0 0.025 1.3 0.0191.6 0.009 1.2 Cal C 0.488 0.034 1.0 0.028 1.4 0.046 3.9 0.011 1.6 Cal D1.95 0.051 1.5 0.029 1.5 0.138 11.8 0.014 2.0 Cal E 7.81 0.112 3.2 0.0392.0 0.121 10.3 0.037 5.3 Cal F 31.3 0.339 9.7 0.103 5.3 1.938 165.10.101 14.2 Cal G 125 1.079 31.0 0.221 11.3 7.245 617.2 0.406 57.0 Cal H500 3.132 89.9 0.667 34.0 21.285 1813.1 1.594 223.7 GFAP Hytest vendorCal A 0 0.015 0.025 0.008 0.008 Cal B 0.122 0.020 1.3 0.024 0.9 0.0202.5 0.013 1.5 Cal C 0.488 0.021 1.4 0.024 0.9 0.034 4.2 0.010 1.2 Cal D1.95 0.022 1.5 0.026 1.0 0.076 9.4 0.011 1.3 Cal E 7.81 0.020 1.3 0.0240.9 0.281 34.4 0.012 1.5 Cal F 31.3 0.015 1.0 0.025 1.0 1.123 137.60.012 1.5 Cal G 125 0.020 1.3 0.025 1.0 4.672 572.7 0.012 1.5 Cal H 5000.023 1.5 0.032 1.3 14.939 1831.5 0.013 1.6

Example 5. The following non-limiting example describes thecross-reactivity and/or non-specific binding of certain UCH L1 cloneswith NF-L capture beads. UCH L1 clones 2D3 and 1016 produced arelatively high non-specific binding signal and were incompatible withthe multiplex format as shown in Table B-1. Conversely, UCH L1 clones10D1 and 1033 did not produce a detectable non-specific signal as shownin Table B2.

TABLE B-1 UCHL1 antibody pair 2D3-1016 causing crosstalk Antigen TauAntigen NF-L Beads Tau NF-L Beads Tau NF-L Detector Det Mix Det MixDetector Det Mix Det Mix Calibrator pg/mL AEB S/B AEB S/B Calibratorpg/mL AEB S/B AEB S/B Cal A 0 0.031 0.400 Cal A 0 0.034 0.548 Cal B0.024 0.039 1.3 0.436 1.1 Cal B 0.014 0.037 1.1 0.522 1.0 Cal C 0.0980.051 1.7 0.444 1.1 Cal C 0.055 0.030 0.9 0.542 1.0 Cal D 0.391 0.1063.5 0.418 1.0 Cal D 0.218 0.033 1.0 0.608 1.1 Cal E 1.56 0.302 9.8 0.4491.1 Cal E 0.87 0.028 0.8 0.717 1.3 Cal F 6.25 0.461 15.0 0.461 1.2 Cal F3.49 0.029 0.9 #DIV/0! #DIV/0! Cal G 25.0 2.882 93.9 0.489 1.2 Cal G14.0 0.031 0.9 #DIV/0! #DIV/0! Cal H 100 9.314 303.3 0.587 1.5 Cal H55.9 0.032 1.0 #DIV/0! #DIV/0! Antigen GFAP Vendor A Antigen UCHL1Vendor B Beads Tau NF-L Beads Tau NF-L Detector Det Mix Det Mix DetectorDet Mix Det Mix Calibrator pg/mL AEB S/B AEB S/B Calibrator pg/mL AEBS/B AEB S/B Cal A 0 0.035 0.515 Cal A 0 0.029 0.516 Cal B 0.122 0.0331.0 0.468 0.9 Cal B 1.22 0.032 1.1 0.539 1.0 Cal C 0.488 0.034 1.0 0.4770.9 Cal C 4.88 0.033 1.2 0.511 1.0 Cal D 1.95 0.051 1.5 0.499 1.0 Cal D19.5 0.034 1.2 0.494 1.0 Cal E 7.81 0.112 3.2 0.570 1.1 Cal E 78.1 0.0351.2 0.515 1.0 Cal F 31.3 0.339 9.7 0.589 1.1 Cal F 313 0.032 1.1 0.4330.8 Cal G 125 1.079 31.0 0.726 1.4 Cal G 1250 0.032 1.1 0.458 0.9 Cal H500 3.132 89.9 #DIV/0! #DIV/0! Cal H 5000 0.041 1.4 0.471 0.9

TABLE B2 UCHL1 antibody pair 1001-1033 had minimal crosstalk AntigenBeads Tau Ab42 GFAP UCHL1 Detector Det Mix Det Mix Det Mix Det MixCalibrator pg/mL AEB S/B AEB S/B AEB S/B AEB S/B Tau Cal A 0 0.011 0.0280.014 0.010 Cal B 0.024 0.013 1.2 0.028 1.0 0.015 1.0 0.011 1.1 Cal C0.098 0.026 2.4 0.026 0.9 0.012 0.8 0.010 0.9 Cal D 0.391 0.050 4.70.026 0.9 0.013 0.9 0.014 1.3 Cal E 1.56 0.178 16.5 0.028 1.0 0.015 1.10.012 1.1 Cal F 6.25 0.673 62.7 0.025 0.9 0.017 1.2 0.018 1.7 Cal G 25.02.184 203.6 0.031 1.1 0.030 2.1 0.036 3.5 Cal H 100 8.638 805.0 0.0371.3 0.076 5.3 0.109 10.5 Ab42 Cal A 0 0.015 0.029 0.012 0.010 Cal B0.014 0.012 0.8 0.029 1.0 0.013 1.0 0.015 1.4 Cal C 0.055 0.016 1.10.051 1.8 0.014 1.1 0.012 1.1 Cal D 0.218 0.012 0.8 0.104 3.6 0.014 1.20.014 1.4 Cal E 0.87 0.017 1.1 0.310 10.7 0.012 1.0 0.011 1.1 Cal F 3.490.015 1.0 1.187 40.8 0.014 1.1 0.013 1.3 Cal G 14.0 0.014 1.0 3.648125.5 0.014 1.1 0.014 1.3 Cal H 56 0.017 1.2 14.216 489.1 0.013 1.10.013 1.3 UCHL Cal A 0 0.029 0.029 0.014 0.013 Cal B 1.22 0.032 1.10.033 1.1 0.014 1.0 0.018 1.4 Cal C 4.88 0.033 1.2 0.030 1.0 0.016 1.10.025 2.0 Cal D 19.5 0.034 1.2 0.030 1.0 0.014 1.0 0.056 4.4 Cal E 78.10.035 1.2 0.031 1.1 0.014 1.0 0.182 14.5 Cal F 313 0.032 1.1 0.032 1.10.016 1.1 0.638 50.8 Cal G 1250 0.032 1.1 0.032 1.1 0.022 1.6 2.051163.1 Cal H 5000 0.041 1.4 0.040 1.4 0.039 2.7 9.591 762.7 GFAP Cal A 00.015 0.025 0.008 0.008 Cal B 0.122 0.020 1.3 0.024 0.9 0.020 2.5 0.0131.5 Cal C 0.488 0.021 1.4 0.024 0.9 0.034 4.2 0.010 1.2 Cal D 1.95 0.0221.5 0.026 1.0 0.076 9.4 0.011 1.3 Cal E 7.81 0.020 1.3 0.024 0.9 0.28134.4 0.012 1.5 Cal F 31.3 0.015 1.0 0.025 1.0 1.123 137.6 0.012 1.5 CalG 125 0.020 1.3 0.025 1.0 4.672 572.7 0.012 1.5 Cal H 500 0.023 1.50.032 1.3 14.939 1831.5 0.013 1.6

Example 6. The following non-limiting example describes the use ofcertain detergents in the sample diluent. Triton™ X-100 was found to besuitable for both Tau and UCH L1 assays as shown in Table C.

TABLE C UCHL1 Normalized UCHL1 Normal CSF Tau by Tau Zero sample conesample Sample Diluent Detergent signal signal (pg/mL) diluent Samplediluent 1 1% 3-(Decyldimethyl) 0.0030 0.0188 2.58  2% Sample diluent 21% N-Dodecyl 0.0038 84.37 50% Sample diluent 3 0.5% 3-(Decyldimethyl)0.0060 20.75 12% Sample diluent 4 0.5% N-Dodecyl 0.0043 83.84 50% Samplediluent 5 0.5% Triton 0.0069 160.10 95% Sample diluent 6 0.5% Tw200.0078 147.59 88% Sample diluent 7 0.5% ASB14 0.0033 160.43 95% Tausample diluent 1% ASB14 0.0029 168.10 100% 

Example 7. The following non-limiting example describes the use ofblockers in the sample diluent to impart dilution linearity andpredictable spike recovery. Superchemiblock™ produced linear dilutionsand predictable spike recovery as shown in Table D.

TABLE D NF-L Buffer Corrected Recovery Sample ID Spiking Level w/oSuperchemiblock w Superchemiblock Buffer 10 pg/mL 100%  100%  100 pg/mL100%  100%  Serum 1 10 pg/mL 43% 76% 100 pg/mL 34% 74% Serum 2 10 pg/mL61% 92% 100 pg/mL 52% 104%  Average 48% 87% Dilution w/o Superchemiblockw Superchemiblock NF-L Spiked Serum Dilution Linearity 1:4  100% 100%1:8  106%  96% 1:16 139% 112% 1:32 156% 117% 1:64 181% 116%  1:128 212% 99% Average 159% 108% NF-L Endogenous CSF Dilution Linearity 1:4  100%100% 1:8  102% 123% 1:16 112% 115% 1:32 129% 113% 1:64 143% 104%  1:128192% 119%  1:256 301% 127%  1:512 602% 108% Average 210% 114% Tau SpikedSerum Dilution Linearity 1:4  100% 100% 1:8  100%  96% 1:16 115%  89%1:32 152%  82% 1:64 171%  82%  1:128 186%  88%  1:256 217%  82%  1:512236% 102% Average 160%  90%

Example 8. The following non-limiting example describes the effect ofdetection molecule concentration on non-specific binding. Table E showsAEB and S/B for NF-L for different concentrations of UCH L1 detector.The concentration of UCH L1 detection molecules used in a single assay(i.e., 5 pg/mL) and the concentration used in the multiplex assay (i.e.,0.5 pg/mL) were investigated.

TABLE E Plex 4-plex- NFL [UCHL1 Det] 5 ug/mL 0.5 ug/mL Calibrator pg/mLAEB S/B AEB S/B Cal A 0 0.014 0.008 Cal B 1 0.017 1.3 0.016 1.9 Cal C 50.108 7.8 0.100 12.0 Cal D 50 0.864 62.4 0.884 106.1 Cal E 500 9.213664.8 8.874 1065.0

For description of various details associate with this assay, see, forexample, U.S. patent application Ser. No. 12/731,130, filed Mar. 24,2010, published as US-2011-0212848 on Sep. 1, 2011, entitled“ULTRA-SENSITIVE DETECTION OF MOLECULES OR PARTICLES USING BEADS OROTHER CAPTURE OBJECTS,” by Duffy et al.; International PatentApplication No. PCT/US2011/026645, filed Mar. 1, 2011, published as WO2011/109364 on Sep. 9, 2011, entitled “ULTRA-SENSITIVE DETECTION OFMOLECULES OR PARTICLES USING BEADS OR OTHER CAPTURE OBJECTS,” by Duffyet al.; International Patent Application No. PCT/US2011/026657, filedMar. 1, 2011, published as WO 2011/109372 on Sep. 9, 2011, entitled“ULTRA-SENSITIVE DETECTION OF MOLECULES USING DUAL DETECTION METHODS,”by Duffy et al.; U.S. patent application Ser. No. 12/731,135, filed Mar.24, 2010, published as US-2011-0212462 on Sep. 1, 2011, entitled“ULTRA-SENSITIVE DETECTION OF MOLECULES USING DUAL DETECTION METHODS,”by Duffy et al.; International Patent Application No. PCT/US2011/026665,filed Mar. 1, 2011, published as WO 2011/109379 on Sep. 9, 2011,entitled “METHODS AND SYSTEMS FOR EXTENDING DYNAMIC RANGE IN ASSAYS FORTHE DETECTION OF MOLECULES OR PARTICLES,” by Rissin et al.; U.S. patentapplication Ser. No. 12/731,136, filed Mar. 24, 2010, published asUS-2011-0212537 on Sep. 1, 2011, entitled “METHODS AND SYSTEMS FOREXTENDING DYNAMIC RANGE IN ASSAYS FOR THE DETECTION OF MOLECULES ORPARTICLES,” by Duffy et al.; U.S. patent application Ser. No.13/035,472, filed Feb. 25, 2011, published as US 2012-0196774, entitled“SYSTEMS, DEVICES, AND METHODS FOR ULTRA-SENSITIVE DETECTION OFMOLECULES OR PARTICLES,” by Fournier et al.; U.S. patent applicationSer. No. 13/037,987, filed Mar. 1, 2011, published as US-2011-0245097 onOct. 6, 2011, entitled “METHODS AND SYSTEMS FOR EXTENDING DYNAMIC RANGEIN ASSAYS FOR THE DETECTION OF MOLECULES OR PARTICLES,” by Rissin etal.; each herein incorporated by reference.

Examples 9-11: Analysis of plasma samples from patients with mildtraumatic brain injury. A series of assay experiments for NF-L, Tau,GFAP, and UCH L1 were performed on blood samples obtained from donorssuspected for mild TBI and from a control group. Donor characteristicsare shown in Table 9 and results are shown in Tables 10-11. See Gill, J.et al., “Glial fibrillary acidic protein elevations relate toneuroimaging abnormalities after mild TBI,” Neurology 91:15 (Oct. 9,2018) e1385-e1389.

TABLE 9 Donor Characteristics in Examples 9-11 Injury Mechanisms Numberof Demographic Characteristics² Vehicular Struck by Example¹ Donors AgeGCS NSI Male Accident Fall Assault Object Example 9 69 52.0 14.07 15.8065% 40% 21% 17% 13% Example 10 28 46.0 14.42 14.89 60% 46% 32% 25%  7%Example 11 177 41.5 14.33 14.51 57% 54% 28% 18%  9% Comparative 49 48.6n.a. n.a. 61% n.a. n.a. n.a. n.a. Example A ¹Donors in Examples 9-11were participants in Traumatic Head Injury Neuroimaging Classificationstudy (NCT01132937). EDTA blood samples (venous) were collected within48 hours of injury, plasma obtained, and stored at −80° C. Donors inComparative Example A were healthy donors without a history of TBI orneurologic disease. ²Mean age (“Age”), Glasgow Coma Scale (“GCS”),Neurobehavioral Symptom Inventory (“NSI”), and percentage male (“Male”)presented.

TABLE 10 Assay and Imaging Results Assay Results¹ Imaging Results²Example NF-L Tau GFAP UCH L1 MRI CT Example 9 23.20 (11.82-44.78)  2.84(1.86-4.17) 2853 (768.2-5724) 53.62 (30.60-149.5) + + Example 10 17.94(11.68-32.56) 2.32 (1.7-7.33) 2098 (253.7-8382) 42.16 (16.63-96.20) + −Example 11 7.62 (4.56-16.09) 1.76 (1.1-2.88) 266.2 (101.8-857.2) 23.06(13.44-44.06) − − Comparative 4.87 (3.04-7.11)   1.25 (0.71-2.09) 56.18(45.98-69.84) 9.59 (6.01-19.86) n.a. n.a. Example A Statistical x² =80.99 x² = 36.94 x² = 143.8 x² = 45.99 n.a. n.a. Significance³ p <0.00011 p < 0.00011 p < 0.00011 p < 0.00011 ¹Analytes were quantified inplasma samples by multiplex assay using the Simoa (Single MoleculeArray) Neurology 4-plex assay kit (Quanterix, Lexington, MA). Median andinterquartile ranges presented in pg/mL ²MRI and CT performed within 48hours of injury. MRI protocol comprised diffusion-tensor imaging,T2*-weighted imaging, T2-fluid-attenuated inversion recovery (FLAIR),high-resolution 3D-T1, dynamic susceptibility contrastperfusion-weighted imaging, and post-contrast T1 and T2-FLAIR.³Differences between the Examples and Comparative Example werecalculated using the Kruskal-Wallis and the Mann-Whitney tests, andcorrelation analyses were performed using the Spearman rank test, withp-values adjusted for multiple comparisons with the Benjamini-Hochbergprocedure. All tests were 2-tailed; p < 0.05 was considered significant.

TABLE 11 AUC's for Age-Adjusted ROC Curves in Examples 9-11 Donors UsedTrue True Negative Positive AUC ROC Curve Condition Condition NF-L TauGFAP Healthy donors vs. donors Comparative Examples 0.7668 0.6777 0.9283suspected for mild Example A 9-11 traumatic brain injury (control vs.mTBI) Donors with positive vs. negative Examples Example 0.6539 0.65720.7722 computed CT indication of brain injury 10-11 9 (CT⁺ vs. CT⁻)Donors with positive vs. negative Example Examples 0.6628 0.7170 0.8032MRI indication of brain injury 11 9-10 (MRI⁺ vs. MRI⁻) Donors withnegative CT and negative Example Example 0.6399 0.6836 0.7402 MRIindication of brain injury vs. negative 11 10 CT and positive MRIindications of brain injury (CT⁻ MRI⁻ vs. CT⁻ MRI⁺)

Examples 12: Analysis of plasma samples from patients with traumaticbrain injury. A series of assay experiments for NF-L, Tau, GFAP, and UCHL1 were performed on venous blood samples obtained from donors suspectedfor TBI. Donor characteristics are shown in Table 12 and results areshown in Tables 13-14. See Korley, F. K., “Performance Evaluation of aMultiplex Assay for Simultaneous Detection of Four Clinically RelevantTraumatic Brain Injury Biomarkers,” J. Neurotrauma 36 (Jan. 1, 2019)182-187.

TABLE 12 Donor Characteristics in Examples 12-13 Injury Mechanisms Num-Vehi- Pedes- ber Demographic Characteristics² cular trian StruckPost-traumatic Amnesia Exam- of GCS Acci- Vehicular by Sus- Un- ple¹Donors Age 3-8 9-12 13-15 Male dent Accident Fall Assault Object Yespected No known Example 63 39.0   0% 1.6% 98.4% 63.5% 27.0% 6.3% 49.2%12.7% 4.8% 55.6%  1.6% 33.3%  9.5% 12 Example 44 44.0 25.0% 9.1% 65.9%86.4% 39.5% 2.3% 44.2% 14.0% 0.0% 43.2% 18.2% 18.2% 20.5% 13 ¹Donors inExample 12 and Comparative Example B were participants in theTransforming Research and Clinical Knowledge in TBI Pilot (TRACK-TBPilot Study). Venous blood samples were collected within 24 hours ofinjury. Donors in Example 12 showed no intracranial abnormality on headCT (CT⁺) while donors in Comparative Example B showed no intracranialabnormality on head CT (CT⁻). Samples were centrifuged and plasmaaliquots were stored at −80° C. ²Median age (“Age”), Glasgow Coma Scale(“GCS”), and percentage male (“Male”) presented.

TABLE 13 Assay and CT Scan Results Assay Results¹ Example NF-L Tau GFAPUCH L1 CT² Example 12 8.8 (5.6-13.8) 3.5 (2.2-6.5) 540.0 (104.5-1265.9) 38.8 (22.4-62.5) − Example 13 20.4 (15.2-39.1) 11.0 (4.9-17.2) 4406.3(2677.5-13630.7) 120.6 (70.7-226.4) + Statistical p < 0.001 p < 0.001 p< 0.001 p < 0.001 n.a. Significance³ ¹Analytes were quantified in plasmasamples by multiplex assay using the Simoa (Single Molecule Array)Neurology 4-plex assay kit (Quanterix, Lexington, MA). Median andinterquartile ranges presented in pg/mL. Digital assay results wereexamined for normality with distributional plots and the Shapiro-Wilktest. ²Head CT performed within 24 hours of injury. ³The Mann-Whitney Utest was used to assess for between-group differences in the digitalassay results.

TABLE 14 AUC's for Age-Adjusted ROC Curves in Examples 12-13 Donors UsedTrue Negative True Positive AUC ROC Curve Condition Condition NF-L TauGFAP UCH L1 Donors with normal vs. abnormal Example 12 Examples 13 0.840.77 0.88 0.86 CT scans (CT⁻ vs. CT⁺)

Examples 14-16: Analysis of plasma samples from patients with RRMS. Aseries of digital assay experiments for NF-L, Tau, GFAP, and UCH L1 wereperformed on blood samples obtained from donors diagnosed with RRMS.Results are shown in Table 15 and FIGS. 8-11.

TABLE 15 Assay Results in Examples 14-16 Relapsing- remitting NumberMultiple of Assay Results² Sclerosis Example¹ Donors NF-L Tau GFAP UCHL1 Severity³ Example 14 6 7.57 ± 1.43  1.0 ± 0.459 61.9 ± 10.4 10.5 ±1.09 Mild Example 15 6 10.4 ± 1.7  1.09 ± 0.218  135 ± 16.5 12.9 ± 3.75Moderate Example 16 4 23.4 ± 8.87 2.08 ± 0.864  175 ± 20.3 46.0 ± 33.7Severe Comparative 12  5.14 ± 0.623 2.02 ± 0.277 62.2 ± 5.82 8.96 ± 1.93n.a. Example B⁴ ¹EDTA blood samples (venous) were obtained fromBioreclamation. Samples from patients diagnosed with RRMS were activelyreceiving medical treatment. ²Analytes were quantified in plasma samplesby multiplex assay using the Simoa (Single Molecule Array) Neurology4-plex assay kit (Quanterix, Lexington, MA). Mean concentration ispresented in pg/mL. ³RRMS severity was categorized based on aphysician's diagnosis. Mild: Expanded Disability Status Scale (EDSS)scores less than 3; moderate: EDSS scores between 3 and 5; severe: EDSSscores greater than 5. ⁴Donors in Comparative Example B were healthydonors.

Prophetic Examples

Prophetic Examples 17-19: Assay analysis of samples diluted with samplediluent containing added human IgG. A series of assay experiments wouldbe performed on samples at various levels of dilution and with andwithout added human IgG. Diluent compositions are shown in Table 16. Theresults shown in Tables 17-19 would be obtained.

TABLE 16 Diluent Compositions in Example 17-19 Examples 50 mM phosphate,137 mM NaCl, 2.7 mM KCl, 0.02% BSA, 1 mM 17-19 MgCl₂, 0.06% dextrose,0.01% BgG, 5 mM urea, 0.5% Triton ™ X-100, 10 mcg/mL TRU block ™, 50mcg/mL Superchemiblock ™, 5 mg/mL human IgG, and 0.05% ProClin 300 ™ atpH 7.4. Comparative 50 mM phosphate, 137 mM NaCl, 2.7 mM KCl, 0.02% BSA,1 mM Examples MgCl₂, 0.06% dextrose, 0.01% BgG, 5 mM urea, 0.5% C-ETriton ™ X-100, 10 mcg/mL TRU block ™, 50 mcg/mL Superchemiblock ™, and0.05% ProClin 300 ™ at pH 7.4.

TABLE 17 Assay Results for Spiked Samples in Example 171 NF-L Tau GFAPUCH L1 Example Comparative Example Comparative Example ComparativeExample Comparative Matrix² 17 Example C 17 Example C 17 Example C 17Example C Serum 80-120% <70% 80-120% <70% 80-120% <70% 80-120% <70%Plasma 80-120% <70% 80-120% <70% 80-120% <70% 80-120% <70% Plasma80-120% <70% 80-120% <70% 80-120% <70% 80-120% <70% Plasma 80-120% <70%80-120% <70% 80-120% <70% 80-120% <70% Serum 80-120% <70% 80-120% <70%80-120% <70% 80-120% <70% Serum 80-120% <70% 80-120% <70% 80-120% <70%80-120% <70% ¹Samples would be spiked with 100 pg/mL each of NF-L, Tau,GFAP, and UCH L1. Spike recovery is the ratio of observed concentrationfollowing subtraction of endogenous NF-L to expected concentrationexpressed as a percentage. ²Samples would be diluted 4x with the samplediluent indicated in Table 16.

TABLE 18 Assay Results for Unspiked Samples¹ Ratio of Concentration ofIndicated Analyte in Example 18 to Comparative Example D Matrix² NF-LTau GFAP UCH L1 Serum 0.9-1.1 0.9-1.1 0.9-1.1 0.9-1.1 Plasma 0.9-1.10.9-1.1 0.9-1.1 0.9-1.1 Plasma 0.9-1.1 0.9-1.1 0.9-1.1 0.9-1.1 Plasma0.9-1.1 0.9-1.1 0.9-1.1 0.9-1.1 Serum 0.9-1.1 0.9-1.1 0.9-1.1 0.9-1.1Serum 0.9-1.1 0.9-1.1 0.9-1.1 0.9-1.1 ¹Concentration is presented inpg/mL. ²Samples would be diluted 4× with the sample diluent indicated inTable 16.

TABLE 19 Assay Results of Plasma at Different Levels of Dilution¹ Ratioof Observed to Expected Concentration for Each of NF-L, Tau, GFAP, andUCH L1 Based on 4× Dilution Dilution² Example 19 Comparative Example E 4×     100%     100%  8× 100-115% 120-150% 16× 105-130% 150-180% 32×110-140% 190-220% 64× 120-150% 210-240% ¹Spike recovery is the ratio ofobserved concentration following subtraction of endogenous NF-L toexpected concentration expressed as a percentage.

Prophetic Example 20: Multiplex assay calibration experiments. A seriesof calibration solutions with and without added non-analyte proteinswould be assayed for the following biomarkers: NF-L, GFAP, UCH L1, andTau. The results reported in Table 20 would be attained.

TABLE 20 Assay¹ Calibration Results in Example 20 Calibration Solution²Measured concentration NF-L GFAP UCH L1 Tau NF-L GFAP UCH L1 Tau 0 0 0 0LOB LOB LOB LOB 0.5 1 10 0.1 >LOQ >LOQ >LOQ >LOQ 1.5 3 300.3 >LOQ >LOQ >LOQ >LOQ 5 10 100 1 >LOQ >LOQ >LOQ >LOQ 15 30 3003 >LOQ >LOQ >LOQ >LOQ 50 100 1000 10 >LOQ >LOQ >LOQ >LOQ 150 300 300030 >LOQ >LOQ >LOQ >LOQ 450 900 9000 90 >LOQ >LOQ >LOQ >LOQ ¹Separateportions of carboxy-terminated, 2.7 micron-diameter magnetic beads(Agilent Technologies) would be prepared by conjugating with anti-NF-Lmouse monoclonal antibodies, anti-GFAP mouse monoclonal antibodies,anti-UCH L1 mouse monoclonal antibodies, anti-Tau protein mousemonoclonal antibodies, anti-Amyloid beta 42/40 mouse monoclonalantibodies, anti-S100B mouse monoclonal antibodies, and anti-NSE mousemonoclonal antibodies (via carbodiimide coupling), blocking, and mixingthe magnetic beads together. The mixed antibody-coated beads would bediluted in a buffer solution. 100 mcL calibration solution would bemixed with a volume of the bead solution to provide 10⁹ beads andincubated, the beads washed post-incubation, resuspended and incubatedwith biotinylated detector reagent, washed, resuspended and incubatedwith streptavidin-β-galactosidase, loaded into microwell arrays, andNF-L, GFAP, UCH L1, Tau, A beta 40, S100B, and NSE quantified using aQuanterix Simoa HD-1 Analyzer (Quanterix ™, Lexington, Massachusetts).²Calibration solution at indicated concentrations of NF-L, GFAP, UCH L1,and Tau (pg/mL), 50 mM phosphate, 137 mM NaCl, 2.7 mM KCl, 2% BSA, 0.1%10 G Surfactant, 10 mcg/mL TRU block ™, 0.05% ProClin ™ 300, and 5 mMEDTA.

Calibration curves for the Examples would be obtained to express thebiomarker concentrations (pg/mL) as a function of AEB by third-orderpolynomial fitted to the AEB data.

Prophetic examples 21-26: Analysis of plasma samples from patientssuspected of traumatic brain injury. A series of multiplex digitalassays would be conducted on samples obtained from venous blood andAEB's obtained using a Quanterix Simoa HD-1 Analyzer (Quanterix™,Lexington, Mass.). The results reported in Table 21 would be obtained.

TABLE 21 Biomarker Assay Results¹⁻² Prophetic Example Donor SampleTiming NF-L GFAP UCH L1 Tau Example 21 Suspected of mild Within 12 hoursof >LOQ >LOQ >LOQ >LOQ traumatic event Example 22 brain injury  7 dayspost event >LOQ >LOQ >LOQ >LOQ Example 23 14 days postevent >LOQ >LOQ >LOQ >LOQ Example 24 Suspected of Within 12 hoursof >LOQ >LOQ >LOQ >LOQ traumatic event Example 25 brain injury  7 dayspost event >LOQ >LOQ >LOQ >LOQ Example 26 14 days postevent >LOQ >LOQ >LOQ >LOQ ¹Plasma obtained from venous blood samplesobtained at the indicated timing would be diluted in sample diluent andassayed. The sample diluent would comprise 50 mM phosphate, 137 mM NaCl,2.7 mM KCl, 0.02% BSA, 1 mM MgCl₂, 0.06% dextrose, 0.01% BgG, 5 mM urea,0.5% Triton ™ X-100, 10 mcg/mL TRU block ™, 50 mcg/mL Superchemiblock ™,and 0.05% ProClin ™ 300. ²Biomarker concentrations (pg/mL) would beobtained using the calibration curve obtained for Example 20.

Example 27: Assay analysis of samples diluted with sample diluentcontaining added IgG. A series of digital assay experiments wouldperformed on samples diluted with added human IgG, mouse IgG or bovineIgG. Diluent compositions are shown in Table 22. The results shown inTable 23 would be obtained.

TABLE 22 Diluent Compositions in Example 27 Example 27 50 mM Phosphate,137 mM NaCl, 2.7 mM KCl, 0.02% BSA, 1 mM MgCl₂, 0.06% dextrose, 0.01%BgG, 5 mM urea, 0.5% Triton ™ X-100, 10 mcg/mL TRU block ™, 50 mcg/mLSuperchemiblock ™, 5 mg/mL human IgG, and 0.05% ProClin 300 ™ at pH 7.4.Comparative 50 mM Phosphate, 137 mM NaCl, 2.7 mM KCl, 0.02% BSA, 1 mMMgCl₂, 0.06% Example F Dextrose, 0.01% BgG, 5 mM Urea, 0.5% TritonX-100, 10 mcg/mL TRU block ™, 50 mcg/mL Superchemiblock ™, 5 mg/mL mouseIgG, and 0.05% ProClin 300 at pH 7.4 Comparative 50 mM Phosphate, 137 mMNaCl, 2.7 mM KCl, 0.02% BSA, 1 mM MgCl₂, 0.06% Example G Dextrose, 0.01%BgG, 5 mM Urea, 0.5% Triton X-100, 10 mcg/mL TRU block ™, 50 mcg/mLSuperchemiblock ™, 5 mg/mL bovine IgG, and 0.05% ProClin 300 at pH 7.4

TABLE 23 Assay Results for Spiked Samples in Example 27¹ Spike Recoveryfor Each of NF-L, Tau, GFAP, and UCH L1 Each of Comparative ExampleExamples Matrix² 27 F and G Plasma 80-120% <70% Serum 80-120% <70% Serum80-120% <70% ¹Spike recovery is the ratio of observed concentrationfollowing subtraction of endogenous NF-L to expected concentrationexpressed as a percentage. ²Samples would be diluted 4x with the samplediluent indicated in Table 22.

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. It is intendedthat the following claims define the scope of the invention and thatmethods and structures within the scope of these claims and theirequivalents be covered thereby.

What is claimed is:
 1. A kit, comprising: i) a plurality of captureagents configured to separately bind to two or more types of analytesselected from the group consisting of NF-L, GFAP, UCH L1, and Tau; ii) aplurality of detection agents configured to separately bind to theselected two or more types of analytes; iii) a sample diluent comprisinghuman IgG at a concentration of between 1 mg/mL and 10 mg/mL; and iv) aplurality of calibration solutions comprising a plurality ofpredetermined concentrations of the selected two or more types ofanalytes.
 2. The kit of claim 1, wherein the sample diluent furthercomprises at least 15 mcg/mL of at least one heterophile blocking agentexclusive of the human IgG.
 3. The kit of claim 1, wherein the pluralityof calibration solutions are pre-diluted for use to determine acalibration standard curve without further dilution.
 4. The kit of claim3, wherein the two or more types of analytes is four types of analytesconsisting of NF-L, GFAP, UCH L1, and Tau.
 5. The kit of claim 4,wherein the plurality of calibration solutions is between 6 and 10calibration solutions, inclusive of an NF-L-free, GFAP-free, UCHL1-free, and Tau-free control solution.
 6. The kit of claim 5, whereinthe plurality of calibration solutions comprise: i) a first calibrationsolution comprising NF-L at a concentration of at least 0.5 pg/mL, GFAPat a concentration of at least 1 pg/mL, UCH L1 at a concentration of atleast 10 pg/mL, and Tau at a concentration of at least 0.1 pg/mL; ii) asecond calibration solution comprising NF-L at a concentration of atleast 450 pg/mL, GFAP at a concentration of at least 900 pg/mL, UCH L1at a concentration of at least 9000 pg/mL, and Tau at a concentration ofat least 90 pg/mL; and iii) at least a third calibration solutioncomprising NF-L at a concentration of between 10 pg/mL and 450 pg/mL,GFAP at a concentration of between 20 pg/mL and 900 pg/mL, UCH L1 at aconcentration of between 200 pg/mL and 9000 pg/mL, and Tau at aconcentration of between 2 pg/mL and 90 pg/mL.
 7. The kit of claim 1,wherein at least one of the plurality of capture agents comprises aparamagnetic bead configured for use in a multiplex digital immunoassayanalyzer.
 8. The kit of claim 7, wherein the plurality of detectionagents comprise: i) an NF-L detection agent configured to bind to NF-L;ii) a GFAP detection agent configured to bind to GFAP; iii) a UCH L1detection agent configured to bind to UCH L1; and iv) a Tau detectionagent configured to bind to Tau.
 9. A method to detect a neurologicalcondition, comprising: i) diluting a sample of physiological fluid in adiluent to form a diluted sample; ii) performing a multiplex immunoassayon the diluted sample to obtain a plurality of at least four measuredparameters; and iii) obtaining concentration values for NF-L, GFAP, UCHL1, and Tau, comprising: inputting at least four of the plurality of atleast four measured parameters into a multivariate calibration model.10. The method of claim 9, wherein the plurality of the at least fourmeasured parameters comprise signal readings from a multiplex spottedwell immunoassay.
 11. The method of claim 9, wherein the plurality ofthe at least four measured parameters are derived from Poisson and/orGaussian distribution analysis of results of a multiplex digitalimmunoassay.
 12. The method of claim 9, wherein the multivariatecalibration model is derived from results of a series of multiplexcalibration immunoassays.
 13. The method of claim 12, wherein themultivariate calibration model provides a series of comparativeconcentration values for at least at least two of NF-L, GFAP, Tau, andUCH L1 that are between 80% and 140% proportional when the sample ofphysiological fluid is diluted between 4 times and 64 times.
 14. Themethod of claim 13, wherein the multivariate calibration model has R²values of at least 0.95 for two or more of: i) NF-L at a concentrationof between 1 pg/mL and 50 pg/mL; ii) Tau at a concentration of between0.1 pg/mL and 8 pg/mL; iii) GFAP at a concentration of between 5 pg/mLand 100 pg/mL; and iv) UCH L1 at a concentration of between 20 pg/mL and500 pg/mL.
 15. The method of claim 12, wherein the series of multiplexcalibration immunoassays comprise: i) a first calibration assayperformed on a first calibration solution, the first calibrationsolution comprising NF-L at a first NF-L concentration, GFAP at a firstGFAP concentration, UCH L1 at a first UCH L1 concentration, and Tau at afirst Tau concentration; and ii) at least a second calibration assayperformed on an at least second calibration solution, the at leastsecond calibration solution comprising NF-L at an at least second NF-Lconcentration, GFAP at an at least second GFAP concentration, UCH L1 atan at least second UCH L1 concentration, and Tau at an at least secondTau concentration.
 16. A test for a neurological condition, comprising:i) providing a liquid sample derived from a sample of physiologicalfluid; ii) obtaining, via a multiplex immunoassay, concentrations ofNF-L, GFAP, UCH L1, and Tau in the liquid sample; iii) calculating atleast one classification value based on a multivariate classificationmodel using the concentrations as inputs to the multivariateclassification model; and iv) assigning a risk of the neurologicalcondition, comprising: comparing the at least one classification valueto at least one threshold value.
 17. The test of claim 16, wherein themultivariate classification model predicts a traumatic brain injury withan ROC curve having an AUC of at least 0.85.
 18. The test of claim 16,wherein the multivariate classification model predicts a traumatic braininjury with a true positive rate of at least 75% at a false positiverate of less than 25%.
 19. The test of claim 16, wherein the testfurther comprises: obtaining a CT scan result negative for theneurological condition prior to performing the multiplex immunoassay.20. The test of claim 16, wherein the multivariate classification modelcomprises a neural network.